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Dihydroflavonol 4-reductase (DFR) is a NADPH-dependent enzyme converting dihydroflavonols to their corresponding leucoanthocyanidins, which are necessary for the formation of anthocyanins and proanthocyanidins (condensed tannins) in higher plants. The biosynthesis pathway of flavonoids is one of the best characterized metabolic pathways in nature. Most key enzymes in the pathway have been determined and their corresponding genes have been cloned and functionally characterized (Winkel-Shirley, 2001). Subsequently, numerous plant sequences generated from different sequencing projects have been annotated as homologs of flavonoid structural genes. In Arabidopsis, there are at least five genes encoding DFR-like proteins (Yuan et al., 2007). However, TT3 represents the only DFR-encoding gene in Arabidopsis (Winkel-Shirley, 2001) and the tt3 mutants are completely deficient in the accumulation of anthocyanin pigments and condensed tannins (Shirley et al., 1995). Other DFR-like genes have been demonstrated to have different biochemical and physiological functions in Arabidopsis. For example, BAN is a structural gene encoding anthocyanidin reductase, which converts anthocyanidins to their corresponding 2, 3-cis-flavan-3-ols for the biosynthesis of proanthocyanidins (Xie et al., 2003). On the other hand, BEN1 was recently demonstrated to be involved in regulating the concentrations of several brassinosteroids, including typhasterol, castasterone, and brassinolide (Yuan et al., 2007). However, the biochemical and physiological functions of the other DFR-like genes in Arabidopsis remain unknown.
We have previously reported a rice DFR-like gene (OsDFR2), which was identified by a cDNA subtraction strategy using anthers from wild-type (AN-N) and thermosensitive male-sterile (AN S-1) mutant indica rice lines (Yau et al., 2005). In situ hybridizations revealed that the expression of OsDFR2 was anther-specific during early microsporogenesis. In the AN S-1 mutant, OsDFR2 expression was down-regulated fourfold under sterility-inducing conditions. These findings suggested that the OsDFR2-derived metabolites, which may be flavonoids, are likely to be necessary for anther development and male fertility in rice. In fact, a specific flavonoid class, flavonols, has been demonstrated to be essential for pollen germination in plant species such as maize and petunia (Coe et al., 1981; Mo et al., 1992; van der Meer et al., 1992). Mutations in the chalcone synthase gene, which encodes the key branch-point enzyme for flavonoid biosynthesis, resulted in the formation of nonfunctional white pollen in these plants. Flavonols were found to be deficient in the mutant pollen and their fertility was completely restored by exogenous application of flavonols (Mo et al., 1992). In Arabidopsis, however, flavonoids do not appear to be critical for male fertility since the different transparent testa (tt) mutants are completely fertile, producing yellow or pale-brown seeds.
The annotation of OsDFR2 as a DFR-like gene was primarily based on sequence homology and no functional data are available for the rice gene. It has been demonstrated that secondary metabolic enzymes are functionally interchangeable between distantly related plants, such as Arabidopsis and maize (Dong et al., 2001). Thus, the Arabidopsis tt mutants are useful for defining the biochemical functions of structural gene homologs from diverse plant species (Yu et al., 2005; Shih et al., 2006). To this end, we over-expressed OsDFR2 in transgenic Arabidopsis tt3 mutants, but the flavonoid deficiencies were not complemented (C. H. Shih & C. Lo, unpublished), indicating that the encoded rice protein does not function as a typical DFR enzyme. It is interesting to note that OsDFR2 not only shows homology to DFR but also to cinnamoyl-CoA reductases and cinnamoyl alcohol dehydrogenases in plants (Yau et al., 2005). In addition, the rice protein also shares homology with mammalian 3-β-hydroxysteroid dehydrogenase, bacterial cholesterol dehydrogenase, and UDP-galactoase-4-epimerase. Hence, OsDFR2 may represent a NADPH-dependent reductase, dehydrogenase, or epimerase that is involved in some novel metabolic pathways critical for male reproductive development in rice.
We took advantage of the comprehensive collections of publicly available Arabidopsis T-DNA mutants to identify mutations that may be useful for us to understand the biochemical and physiological functions of OsDFR2. Two T-DNA insertion alleles were identified in At4g35420, which is the closest homolog of OsDFR2 in Arabidopsis (63% amino acid sequence identity). During propagation of the homozygous mutant lines for seed collection, we observed drastic reduction of seed formation, prompting us to launch an extensive investigation into At4g35420, which we now designate as DRL1 (dihydroflavonol 4-reductase-like1). Here, we report the characterizations of the two insertion mutants, drl1-1 and drl1-2, which showed impaired pollen development leading to different degrees of male sterility. Histological examinations revealed the degeneration of microspores following their release in the mutant anthers as well as the specific expression of the GUS reporter gene driven by the DRL1 promoter in tapetum and developing microspores. Genetic complementation experiments confirmed that DRL1 is essential for pollen development and male fertility in Arabidopsis. In addition to OsDFR2, there are a number of conserved DRL1 homologs (> 60% sequence identity at amino acid level) identified as expressed sequences derived from anther and floral tissues in diverse flowering plant species, suggesting a conserved function of a DFR-like protein in male fertility.
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Our study revealed that a new DFR-like gene, DRL1, is essential for pollen development and male fertility in Arabidopsis. We demonstrated that DRL1 is an anther-specific gene with expression in tetrads, developing microspores, and tapetum. Previously, molecular analysis of anther development had led to the estimation of c. 3500 anther-specific transcripts in Arabidopsis (Sanders et al., 1999). Complete male sterility in the drl1-2 mutant suggested that there are no redundant genes that can replace the unique function of DRL1 in anthers. In addition, complementation of the mutant phenotypes was achieved by ectopic expression of the DRL1 cDNA driven by the native promoter, but not the 35S promoter, indicating that spatial and temporal specificities of transcription are critical for the proper expression of DRL1 physiological functions. The 35S promoter is commonly used for strong constitutive expression of transgenes in plants, including Arabidopsis. However, there have been reports demonstrating that it is not suitable for expression in certain tissue types, including the tapetum (Plegt & Bino, 1989; van der Meer et al., 1992). Tapetum is a layer of sporophytic cells derived from the secondary parietal cells and it plays important roles in the process of microspore development and maturation (Sanders et al., 1999). It secretes enzyme(s) to degrade callose synthesized in microsporocytes for release of microspores into the anther locules. The tapetum also provides nutrient and cell wall materials for the development of functional pollen. A number of genes encoding transcription factors and signal transduction components essential for tapetal functions have been identified in Arabidopsis, providing evidence for a cascade of regulatory network for different stages of tapetal formation and development.
The phenotypes of our drl1 mutants (degeneration of microspores after tetrad release and complete absence of pollen in mature anthers) are reminiscent of those observed in some male-sterile mutants defective in regulatory genes, such as ms1(Wilson et al., 2001) and ams (Sorensen et al., 2003). Thus, DRL1 may represent a primary target of some anther-specific transcription factors. Recently, a number of microarray experiments have been performed to investigate differential gene expression in male-sterile mutant flowers (Alves-Ferreira et al., 2007; Wijeratne et al., 2007; Xing & Zachgo, 2008). Examination of the publicly available data revealed that DRL1 (At4g35420) expression was down-regulated in flower buds of spl/nzz (Alves-Ferreira et al., 2007; Wijeratne et al., 2007), ems1 (Wijeratne et al., 2007), and roxy1roxy2 (Xing & Zachgo, 2008) mutants. However, the ‘down-regulation’ data observed could simply be explained by the fact that tapetum and microspores, in which DRL1 showed strong expression (Fig. 6), never developed in the spl/nzz and ems1 mutants (Schiefthaler et al., 1999; Yang et al., 1999). The tapetum of roxy1roxy2 mutants showed hypertrophic growth after degeneration of pollen mother cells in the abaxial anther lobes while sporogenous cell formation is impaired in the double mutants (Xing & Zachgo, 2008). SPL/NZZ, EMS1, and ROXY1/2 are considered early anther genes controlling sporogenous cell and tapetum formation and they are all expressed in the tapetum at the pollen mother cell stage (Schiefthaler et al., 1999; Yang et al., 1999; Xing & Zachgo, 2008). On the other hand, MS1 is a PHD-finger family transcription factor essential for postmeiotic tapetal development and pollen wall synthesis (Yang et al., 2007) and may also play a role in regulating programmed cell death in tapetum (Vizcay-Barrena & Wilson, 2006). Surprisingly, examination of two independent microarray datasets revealed that DRL1 expression was differentially up-regulated in the ms1 mutant flower buds (Alves-Ferreira et al., 2007; Yang et al., 2007). While the expression pattern of MS1 appeared to overlap with that of DRL1, the microarray data seemed to suggest that MS1 may instead function as a negative regulator of DRL1 expression. Other regulatory genes that were strongly expressed at similar developmental stages as in DRL1 included AMS encoding a MYC protein (Sorensen et al., 2003) and a few genes encoding R2R3 proteins, namely TDF1 (Zhu et al., in press), AtMYB32 (Preston et al., 2004), and AtMYB103 (Higginson et al., 2003). Further genetic and expression analyses in these mutants are necessary to identify the molecular component(s) that activate the anther-specific expression of DRL1. It is possible that some unknown positive regulators function together with MS1 (which might act as a repressor) to maintain an optimal expression level of DRL1 in anthers.
Our cellular localization experiment demonstrated that DRL1 encodes a soluble cytosolic protein, which may also be localized in the nucleus (Fig. 8). Previously, two Arabidopsis flavonoid enzymes (chalcone synthase and chalcone isomerase) were shown to be localized in nuclei of some cells types, in addition to their usual cytosolic location (Saslowsky et al., 2005). Analysis of the DRL1 protein sequence revealed the putative NADPH binding domain (Fig. 9b), which is conserved in the two flavonoid reductase enzymes DFR and BAN (Yuan et al., 2007). However, since the null mutant affecting chalcone synthase is fertile with functional pollen (Burbulis et al., 1996), the male fertility-associated DRL1 is unlikely to be involved in the biosynthesis of flavonoids. Consistent with this, the closely related rice homolog OsDFR2 was unable to complement the tt3 (DFR gene) mutations in Arabidopsis (C. H. Shih & C. Lo, unpublished ). Both DRL1 and OsDFR2 can be regarded as members of the mammalian 3β-hydroxysteroid/plant DFR superfamily, which also includes some bacterial and mammalian enzymes (Yau et al., 2005). The primary metabolic enzyme UDP-galactose-4-epimerase has been suggested to be a putative common ancestor for this family (Baker & Blasco, 1992). Hence, DRL1 may encode a reductase, an epimerase, or a dehydrogenase which functions in anthers to produce some novel metabolites essential for pollen development and male fertility.
The degeneration of drl1 mutant microspores was preceded by the rupture of their abnormal walls (Fig. 3). The formation of pollen wall requires the coordinated metabolic activities in both tapetum and microspores. Since the heterozygous drl1 mutants are indistinguishable from the wild type in terms of fertility and anther development, pollen abortion in the homozygous mutants is unlikely to be caused by defects in gametophytic gene expression. Instead, the tapetal expression of DRL1 at the microspore release stage may play an important role in wall formation in the developing pollen. Primexine, the footprint of the future exine of the pollen wall, is formed at the time of tetrad formation. During microspore release, sporopollenin, the main component of exine, is secreted by the tapetum and deposited on the microspores following the primexine pattern (Paxson-Sowders et al., 1997; Piffanelli et al., 1998). Sporopollenin is a biopolymer of lipid and phenylpropanoid (p-coumaric and ferulic acid) derivatives. So far only Male Sterility2 (MS2) and Faceless Pollen1 (FLP1) have been considered as candidate genes for sporopollenin biosynthesis (Ito et al., 2007). MS2, encoding a putative fatty acyl reductase, was demonstrated to be essential for exine formation (Aarts et al., 1997). Interestingly, MS2 expression was also up-regulated in most stages of the ms1 mutant flower buds (microarray data in Alves-Ferreira et al., 2007), suggesting that MS2 and DRL1 expression may be co-regulated. On the other hand, FLP1 encodes a putative lipid transfer protein that is involved in the formation of both exine and pollen coat (Ariizumi et al., 2003). The biochemistry of sporopollenin formation is still largely unknown, and whether DRL1 is involved in this process remains to be elucidated. The phenylpropanoid pathway enzymes cinnamoyl-CoA reductase and cinnamoyl alcohol dehydrogenase are also members of the mammalian 3β-hydroxysteroid/plant DFR superfamily (Lacombe et al., 1997). Hence, DRL1 may have a role in the synthesis of phenylpropanoid or aromatic precursors, which are released from tapetum to the anther locule for sporopollenin formation in the developing microspores.
Male sterility in plants is a useful trait for different agricultural applications, such as extension of flower shelf-life and production of hybrid seeds (Mitsuda et al., 2006). In addition, generation of genetically modified crops with controlled male sterility is highly desirable as a means for gene containment (Daniell, 2002). A major concern with regard to growing transgenic crops in the open field is the uncontrolled gene flow by cross-pollination of related wild species, leading to generation of ‘superweeds’ as well as disturbance of ecosystem. The occurrence of highly conserved anther- and floral-expressed DRL1 homologs in plant species of diverse families (Poaceae, Asteraceae, Rannuculaceae, Solanaceae) strongly suggested the presence of a common biochemical pathway (or metabolite) essential for pollen development. Hence, DRL1 may be exploited as a useful molecular target for manipulation of male sterility in flowering plants. We are currently investigating the differential metabolite profiles of the drl1 mutant flowers to gain an insight into the biochemical functions of this highly conserved gene which is indispensable for male fertility in Arabidopsis.