|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2006) 30, 583591 (Printed in Great Britain)
Effects of chilling on male gametophyte development in rice
E.A. Mamunab*, S. Alfredab, L.C. Cantrillc, R.L. Overallc and B.G. Suttonab
aCooperative Research Centre for Sustainable Rice Production, The University of Sydney, NSW 2006, Australia
bFaculty of Agriculture, Food and Natural Resources, The University of Sydney, NSW 2006, Australia
cSchool of Biological Sciences, The University of Sydney, NSW 2006, Australia
Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic anthers. Anthers observed during tetrad release also have callose (1,3-β-glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity.
Keywords: Anther, Callose, Carbohydrate metabolism, Chilling, Microspores wall, Rice, RNA in situ hybridisation, OsMST8, Starch accumulation, Ultrastructure.
*Corresponding author. The University of Sydney, Faculty of Agriculture, Food and Natural Resources, John Wooley Building A20, Sydney, NSW 2006, Australia. Tel.: +61 2 93512939; fax: +61 2 93514172.
Chilling detrimentally affects flower induction, pollen production and in some sensitive species it causes male sterility (Satake, 1976; Reyes et al., 2003; Imin et al., 2006; Oliver et al., 2005). The social and economic consequences of chilling and other environmental stresses during reproductive development are particularly important in grain crops like rice and wheat, because the products of their sexual reproduction provide the primary staple for most of humanity.
Over the last 40 years, scientists in Japan have started to search for the most sensitive stage of chilling in rice (Satake and Hayase, 1970; Nishiyama, 1984, 1997). They identified that for rice, the booting stage is the most sensitive period for chilling damage (Takeoka et al., 1992). The period of greatest chilling sensitivity during anther development is the early microspore phase when microspores are released from the tetrads. The beginning of meiosis has also been mentioned as a secondary sensitive period of pollen development to low temperature (Satake, 1976; Takeoka et al., 1992).
Cellular alteration of microspore development in rice anthers under chilling has been described by Nishiyama, a pioneer of research in this area. Much of his work focussed on tapetal morphology under chilling conditions (Nishiyama, 1984, 1997). The morphological changes were mostly associated with dilatation, which Nishiyama described as tapetal hypertrophy. Other researchers went on to classify hypertrophy into different types (Satake, 1976). However, no work to date has been published that demonstrates tapetal hypertrophy in living anthers, so it is unclear whether it is a cellular symptom of chilling stress, or an artefact. Moreover, apart from some recent observations (Truernit et al., 1999), little work has been done on the processes that lead to hypertrophy. In fact, little detailed work has been carried out on any of the cellular features of chilling injury in rice anthers. Satake, another pioneer in this field, published a large body of work during the 1970s and 1980s, yet this work seems to have stopped at the level of whole plant physiology with only a small consideration given to cellular symptoms (Satake, 1976). Ito (1978) and Satake (1976) also gave some consideration to metabolic irregularities in chilled anthers.
Microspore wall formation is also critical under stress conditions. Water deficit inhibits proper microspore wall formation in wheat and rice (Saini et al., 1984; Lalonde et al., 1997a,b) that is associated with carbohydrate metabolism in anthers (Saini and Westgate, 2000). A similar phenomenon of perturbed carbohydrate metabolism has been reported recently in rice anthers under chilling stress (Oliver et al., 2005), however, cellular symptoms of microspore wall formation have not been reported as yet. Callose (1,3-β-glucan), a special wall around the microspore mother cell, dyads and tetrads, plays an important role in microspore wall formation (Mascarenhas, 1975; Pacini, 1994). Premature callose breakdown in mutant tobacco results in male sterility (Worrall et al., 1992). Just before meiosis, callose deposition isolates the microspore mother cells from the tapetum (Mamun et al., 2005b). Moreover, symplastic connections through plasmodesmata between tapetum and other cell layers are not observed at or after meiosis (Mamun et al., 2005b). It is possible that an alternative appoplastic sugar supply pathway is active in the anther at this situation (Truernit et al., 1999), which involves invertase and monosaccharide transporters (MST).
In situ hybridisation and RT PCR gene expression studies in rice revealed that cell wall-bound anther specific invertase gene OsINV4 (accession no. AY220486) is repressed by cold treatment specifically at the young microspore stage (Oliver et al., 2005). A male gametophyte-specific monosaccharide gene OsMST8 (accession no. AY822464) in rice has also been isolated and characterised. It is suggested that this gene plays a critical role in transporting monosaccharides, which are cleavage products of sucrose by invertase, as well as utilising callose breakdown products for microspore development.
To further identify critical biochemical processes, this work documents alteration of cellular organisation during microspore development and the critical role of OsMST8 genes in chilling-induced rice anthers.
2 Materials and methods
2.1 Plant material
Oryza sativa L. cv. Doongara, an Australian rice cultivar that is very low temperature sensitive was selected and grown in soil and hydroponic culture medium as described in Mamun et al. (2005a).
To induce 4 nights' chilling during the early microspore phase, plants at auricle distance approximately −30 to −10
2.2 Electron microscopy and immunogold labelling of 1,3-β-glucan
Anthers were fixed according to Mamun et al. (2005a). At least 100 blocks of normal and chilling-induced anthers were selected and used for light and electron microscopy examination. Transverse ultra-thin sections were collected on coated (grid coating pen; Coat-Quick ‘G’ Daido Sangyo Co., Ltd., Japan) grids and stained with saturated uranyl acetate and Reynold's lead citrate. Sections were observed with a Zeiss 902 electron microscope at 80
2.3 RNA in situ hybridisation
Excised whole florets from normal and chilling-induced rice plants were cut open at the tip. Florets were fixed, embedded, pre-hybridised according to the methods as described in Oliver et al. (2005). Both antisense and sense RNA probes were synthesised by T7 polymerase transcription of the OsMST8 cDNA (kindly provided by Dr. Rudy Dolferus, CSIRO PI, Australia) using a SP6/T7 digoxigenin RNA labelling kit (Roche, Manheim) according to the manufacturer's instructions. Hybridisation and detection of hybrids were performed according to Doughty et al. (1998). Sections were then dehydrated through an ethanol series and washed twice for 2
3.1 Changes in microspores and tapetum cells
After four nights' chilling, severe alterations were seen in the anther layers and microspores. Transverse section of an anther under light microscope indicated that well-shaped vacuolated microspores and other anther layers were seen in the microsporangia in normal anther (Fig. 1a), however, chilling stress altered the whole anther lobe (Fig. 1b). Tapetum and microspores were distorted and outer epidermal layers showed deformation (Fig. 1b). In normal anther development following the microspore release stage, the spherical microspores were seen to be free-floating in the locule and had acquired a conspicuous wall patterning (Fig. 2a). The tapetum at this stage showed dense cytoplasmic contents and numerous well-developed orbicules. In chilling-induced anthers harvested during the free microspore stage, microspores showed clearly visible lesions in development and these were mostly focused around the microspore wall (Fig. 2b,c) but lesions like distortion and deformation were also seen (Fig. 2b). Tapetum cells were severely altered. Distorted tapetal cells along the locule face contained vacuoles of different sizes (Fig. 2b).
Light microscope image of transverse sections of microsporangia showing overview of anther at vacuolated stage. (a) Anther growing in normal temperature comprise spherical microspores and well-shaped anther cell layers. (b) Chilling stress drastically altered the shape and development of the microspores and other cell layers. ep – epidermis; en – endothecium; t – tapetum; mi – microspore.
Part of microsporangia of normal and chilling-induced anthers during free microspore stage. (a) Microsporangium of a normal anther. The spherical microspores acquiring conspicuous wall patterning are free-floating in the anther locule. Cytoplasm of the tapetum is dense and the plasma membrane is decorated with numerous well-developed orbicules. (b) Microsporangium of a cold-treated anther. Microspores show deformation and a lack of wall sculpturing. The cell wall of the tapetum facing the locular cavity has disintegrated and distorted. Arrows show vacuoles of different sizes. (c) Microspore of chilling-induced anther is free floated in the anther locule, however, showing lack of wall sculpturing. (d) Microspore wall at free microspores of a normal anther shows distinctive sculpturing which comprises foot layer, tectum and bacula. (e) Chilling-interrupted microspore wall formation. Only a few sporopollenin (arrows) are seen to be deposited in the microspore wall. (f) Chilling-stressed anther showing hypertrophy. The tapetal cytoplasm is spilled out into the locular cavity and occupies almost the whole cavity. (g) A living anther theca under light microscope showing tapetal hypertrophy (arrows) after chilling. Arrowheads show the tapetal cells. mi – microspore; mw – microspore wall; t – tapetum; en – endothecium; ml – middle layer; chl – chloroplast; l – locule; n – nuclei; f – foot layer; tc – tectum.
Anthers harvested during the free microspore stage suggested that cold drastically altered microspore wall formation (Fig. 2c), however, microspores were characterised by rich cytoplasmic contents but devoid of proper sporopollenin inclusions (Fig. 2c,e). The microspore wall at the free microspore stage was well sculptured and comprised several layers, namely tectum, bacula and foot (Fig. 2d). Micro-channels were seen in the tectum and foot layers (Fig. 2d). The microspore wall was not properly formed in chilling-treated anthers (Fig. 2c,e). The microspore wall was devoid of any fine sculpting. Only a small quantity of sporopollenin had been deposited in a seemingly random way on the chilling-treated microspore wall (Fig. 2e). Other severe lesions of chilling were tapetal hypertrophy (Fig. 2f). Although observed rarely, it appeared that the cytoplasmic contents of the tapetal cells of chilling-induced anthers had spilled out into the locular space (Fig. 2f,g). Observation of hypertrophy in light microscope images (Fig. 2g) of living anthers showed that this phenomenon was not an artefact of preparation for TEM.
3.2 Excessive starch accumulation in the endothecium
Under normal temperatures during the post-meiotic stage, particularly soon after the free microspore stage, endothecium cells contained several chloroplasts along with other organelles (Fig. 3a). However, these chloroplasts consisted of very little internal membrane organisation with very small starch granules and were elongated (Fig. 3a,c). Chloroplasts containing starch granules had a shaded region around the granule indicating that these starch granules were degrading (Fig. 3c). The endothecium cell layers observed after chilling also showed several chloroplasts at the free microspore stage (Fig. 3b). However, these chloroplasts contained an increase in the size and number of starch granules (Fig. 3b,d). Concomitant with starch accumulation, loss of membrane details suggested degeneration of thylakoids and grana as well as the outer membranes in these chloroplasts (Fig. 3b,d).
Development of chloroplasts in the endothecium of normal and chilling-induced anthers. (a) Endothecium cell of an anther develops in normal temperature. This cell shows numerous chloroplasts which contain no or small amount of starch. (b) Endothecium cell from a chilling-induced anthers show numerous chloroplasts, however, they have large starch granules. (c) High magnification image of chloroplasts of an endothecium cell just after free microspore stage become elongated and contain very little starch. However, the chloroplast containing starch granules has regions with very little internal membrane organisation (arrowheads). The starch granule has a shaded region in the outer part of the granule (arrows). (d) Chilling has resulted in increased the size of starch grains and very few granal stacks in the chloroplast of an endothecium. It seems that membranes of the chloroplasts are degenerating (arrows). en – endothecium; chl – chloroplasts; ml – middle layer; t – tapetum; s – starch granule.
3.3 Premature callose dissolution
During the dyad stage in normal anthers, callose was deposited around the dyads and also in the cell plate between two dyad cells (Fig. 4a). Immunogold labelling showed a thick callose labelling at the outside of the dyad (Fig. 4a). However, chilling-induced anthers showed sparse callose in dyad cells (Fig. 4b). Very few gold particles were seen randomly at the outside of the dyad cells (Fig. 4b). In tetrad cells under normal temperatures, immunogold labelling of callose was seen between the four tetrad microspores, however, labelling was very low at the outer wall of these developing microspores (Mamun et al., 2005b). In tetrad microspores from the chilling-induced anthers, callose was rarely observed (Fig. 4c). This contrasts conspicuously with the large number of gold particles seen at normal temperatures at the same stage (greater than 150 gold particles per similar section of wall) (Mamun et al., 2005b). This indicates that callose has virtually degraded around the developing microspores earlier than expected, something that is not observed in normal anthers. Walls of the tetrad microspores of normal anthers showed distinctive wall patterning (Mamun et al., 2005b), however, microspore walls were devoid of any sculpturing (Fig. 4d) in the tetrad microspores from chilling-treated anthers.
Callose distribution in the developing microspore in normal and chilled dyad and treated rice anthers. (a) Profuse numbers of gold particles around the dyad and also in the cell plate of newly formed dyad cells indicates normal callose during dyad formation. (b) Very sparse immunogold labelling (arrowheads) is seen around the dyads indicating early degradation of callose by chilling. (c) Immunogold labelling of callose in chilling-treated tetrad microspores displays a very low level of callose between the tetrad microspore. No sign of typical wall sculpturing is around the newly formed tetrad microspores. Arrowheads show the callose in the central part of a tetrad. Only few (2–3) gold particles indicate very low level of callose. Arrows show poorly developed microspore walls.
3.4 OsMST8 mRNA in situ hybridisation
Chilling treatment at the microspore development stage repressed OsMST8 gene expression. Anthers from plants growing in normal temperatures at the free microspore stage showed OsMST8 gene expression in the tapetum, microspores and vascular bundle (Fig. 5a). However, anthers at a similar stage after 4 days chilling did not show repressed OsMST8 gene expression in the anther layers and vascular bundles (Fig. 5b).
mRNA in situ hybridisation study of OsMST8 in normal and chilling-induced rice anthers at free microspore stage. Transverse sections of (a) normal and (b) chilling induced anthers were labelled with digoxygenin-labelled antisense OsMST8 probe. Transverse section (a) showed positive signals of OsMST8 mRNA (black blue color) in tapetum, microspore and vascular bundles while signal was repressed in different layers in chilling-treated anthers (b). t – tapetum; vb – vascular bundle; mi – microspore.
4.1 Changes in the tapetal cell
The tapetum provides nutrients and structural components for the developing microspores (Pacini, 1994; Raghavan, 1997). Natural male sterility has often been linked to tapetal dysfunction (Loukides et al., 1995; Conicella et al., 1997). Thus it has been believed for a long time that tapetal dysfunction is the key process for chilling-induced male sterility in rice (Nishiyama, 1984, 1997). Similar vacuolations and hypertrophy as seen in the chilling-treated rice anthers associated with male sterility are also seen during meiotic stage water deficit in wheat (Lalonde et al., 1997a), in transgenic tobacco (Worrall et al., 1992; Tsuchiya et al., 1995) and in several cytoplasmic and genic male sterile lines in other species (Loukides et al., 1995; Jin et al., 1997; Hernould et al., 1998).
Nishiyama (1984) has identified tapetal dilatation as a consequence of increasing sugar concentration and turgor pressure in the tapetum. In recent years, this phenomenon has been described more cautiously. Worrall et al. (1992) have proposed that the abnormal increase in volume and vacuolation of the male sterile tapetal cells might be caused by retention in the tapetal cells of materials that would normally be utilised by the developing microspores or might be due to reabsorption of sugars arising from the premature degradation of the callose wall. However, Truernit et al. (1999) have suggested that the hypertrophy of tapetal cells is more likely to occur due to reabsorption of sugars arising from premature callose dissolution. In the chilling-treated rice anthers, premature callose dissolution happens at the same time as inhibition of early microspore wall biosynthesis, so rather than being utilised for microspore wall development, the premature callose breakdown products are most likely reabsorbed by the tapetal cells, which then show hypertrophy or unusual vacuolation.
Another explanation for tapetal vacuolations and hypertrophy might come from problems with sugar movement and utilisation in the tapetum. Although apoplastic invertase is down-regulated in anthers in chilling-treated plants, just as after water stress (Dorion et al., 1996; Sheoran and Saini, 1996; Koonjul et al., 2005), a similar problem might also occur with vacuolar invertase. Sucrose might be stored in vacuoles (Dubinina et al., 2001) in the tapetum if the enzyme is unable to cleave it into monosaccharides for export into the cytosol. Alternatively, mitochondria in tapetal cells might be dysfunctional after chilling and cannot utilise assimilates at the normal rate for energy production. Again, vacuoles might appear as storage organelles for excess unused sucrose. Hypertrophy might be an extreme manifestation of this excess sugar storage in the anther.
4.2 Effects of premature callose dissolution on microspore wall formation
Callose, a 1,3-β-glucan, develops around the meiocytes during early meiosis I and continues until completion of the second meiotic division. A thick callose wall is seen around the meiocyte before meiosis occurs and separates the meiocytes from the rest of the anther cell layers (Mamun et al., 2005a,b). At the early tetrad stage just after second meiosis, it starts to dissolve near the tapetal cell (Mamun et al., 2005b) by a tapetally-secreted callase, a 1,3-β-glucanse (Steiglitz, 1977). Both conventional aniline blue staining and electron microscopy immunogold localisation indicated that callose is a significant component of the tetrad division walls separating tetrad microspores from each other (Mamun et al., 2005b). After the callose is dissolved from and between the microspores, the cells are released into the locule. Mis-timing of callase expression initiates callose wall degradation earlier or later, and has been attributed as a possible cause of male sterility in many male sterile and mutant plants (Izhar and Frankel, 1971; Worrall et al., 1992; Tsuchiya et al., 1995; Jin et al., 1997). There is no report on stress-induced callose expression in the anthers in particular, during chilling in cereal anthers. In chilling-treated rice anthers, callose was suddenly reduced around the dyads and at the early tetrad stage, almost no callose was seen between the tetrads of microspores, unlike tetrads of anthers grown in normal temperatures. This phenomenon might reveal premature callose degradation resembling the cytoplasmic male sterile or mutant plants of other species.
Concomitant with premature callose degradation, chilled rice anthers showed aberrant microspore wall formation. There are significant consequences associated with premature callose degradation and changes of microspore wall formation. Worrall et al. (1992) and Tsuchiya et al. (1995) have shown significant alteration of microspore wall formation as a consequence of premature callose wall degradation in male sterile transgenic tobacco, which resembles the chilling-treated rice anthers. Secretion of a recombinant vacuolar 1,3-β-glucanse from the tapetum of transgenic tobacco plants prior to the appearance of normal callase activity caused drastic inhibition of the exine template (primexine) and subsequent disruption of exine formation (Worrall et al., 1992). Generally, the callose wall is thought to act as a mould for microspore wall formation (Waterkeyn and Beinfait, 1970). In contrast, there are a few species where exine patterning occurs without callose deposition (Periasamy and Amalathas, 1991) or exine forms after microspores are released from tetrads (Christensen et al., 1972).
Truernit et al. (1999) have proposed a plasma membrane-localised monosaccharide transporter, AtSTP2 gene in Arabidopsis, which is only expressed when callose in the tetrads is degraded. A similar transporter, OsMST8 has been identified in rice anthers and showed down-regulation after chilling. This supports the possibility of callose being a source of glucose. It is plausible that in addition to other monosaccharides originating from other wall components or even from the degenerating tapetal cells, OsMST8 or AtSTP2 in the developing pollen would take up the products of callose degradation from the extracellular space for further utilisation. The possibility of such activities is appealing since in rice and other species prior to the meiotic divisions of meiocytes the male gametophyte becomes symplastically isolated from the surrounding sporophytic tissue (Clement and Audran, 1995) and then assimilates needed for gametophytic development have to be taken up by plasma membrane-localised transporters in the developing pollen grain (Truernit et al., 1996; Truernit et al., 1999).
4.3 Starch accumulation and carbohydrate metabolism
Low temperatures induce a substantial amount of starch accumulation in the plastids of rice anthers, however, pollen grains show starch depletion and reduced viability (Oliver et al., 2005). Causes of this are not clear, but there are a whole variety of steps in the carbohydrate cycle in the anther where metabolism or storage of sugar might be upset by chilling. A cell wall-bound acid invertase (OsINV4), which breaks sucrose to fructose and glucose in rice anthers, is poorly expressed or down-regulated by chilling treatment (Oliver et al., 2005). In situ hybridisation results showed that high levels of OsINV4 gene expression were seen in the vascular bundle, endothecium and tapetum during early microspore stage just after microspores were released from tetrads. However, this gene expression was not detected in any of the anther walls and connective tissues during the same stage of development in chilling-treated plants (Oliver et al., 2005). Furthermore, OsMST8 was also repressed by chilling. Repression of both OsINV4 and OsMST8 most likely lead to blockage of sugar supply to the tapetum and pollen grain at a critical point of anther development and help in starch accumulation at plastids in the anther wall.
Additionally, symplastic transport is restricted at the endothecium/middle layer interface in chilled-induced plants. Starch accumulation in the endothecium could therefore be a mechanism to reduce the levels of unused sucrose as it accumulates in the outer cell layers of the anther. Indeed, sugar accumulation in anther tissue has been clearly demonstrated as a characteristic of stresses that lead to male sterility (Saini and Westgate, 2000; Oliver et al., 2005). The role of anther chloroplasts as starch storing buffers to control cellular sugar level has also been demonstrated before in anthers cultured under high sugar concentrations (Clement and Audran, 1995). In addition there are some other possible processes that might result in such a starch accumulation e.g. starch degradative enzymes (e.g. α- and β-amylase and phosphorylase) and sucrose synthase (SucS), which could be altered by chilling, leading to a build up of sugars in these cells; or chilling might decrease the energy demand of the meiocytes or tapetal cells leading to an over-supply of sugars in the anther wall cell layers.
Lalonde et al. (1997a,b) observed that water deficit in wheat anthers increased starch accumulation in the connective tissues at early microsporogenesis. They speculated that this arose from a decrease in carbon utilisation. Activities of soluble acid invertase and cell wall-bound invertases in wheat and rice anthers of water deficit plants decline dramatically, resulting from inhibited gene expression and do not recover even after plants are rehydrated (Koonjul et al., 2005). These attributions of the inhibited invertase activities have been corroborated by another report in tobacco. After meiosis in tobacco, an extracellular invertase, designated as Nin88 (Goetz et al., 2001) is expressed in the tapetal cells and follows a distinct expression pattern during pollen development. Tissue-specific antisense inhibition of Nin88 under control of the corresponding promoter reduces apoplastic invertase activity leading to a developmental arrest of the symplastically isolated pollen at the unicellular microspore stage, thus inducing male sterility (Goetz et al., 2001).
Generally, invertase maintains sucrose concentration gradients between source and sink organs (Sturm et al., 1995; Kim et al., 2000). On the other hand, the sugar level in plant cells modulates the expression of genes involved in carbohydrate metabolism (Koch, 1996). The whole sugar regulation process is thus tightly controlled, with the levels of one component directly affecting the levels of all the others. Thus, starch accumulation in chilling-induced anther walls is a phenomenon of whole sugar regulation in the anther.
The accumulation of abnormal quantities of starch also suggests disruption of the assimilated supply from endothecium cells towards other cell layers and mostly to the growing microspores in the chilled rice anthers. Thus, they are depleted of starch (Oliver et al., 2005). Interrupted symplastic transport pathway in chilling-stressed anthers could restrict carbon transport towards the locule, increase the concentrations of sugars in the outer cell layers and stimulate starch accumulation in the plastids of anther wall tissues.
This research was supported by grants from the Cooperative Research Centre for Sustainable Rice Production (established and supported under the Australian Government's Cooperative Research centres Program), Australia. The authors wish to thank Dr. Rudy Dolferus at CSIRO Plant Industry, Canberra for providing us with the OsMST8 cDNA and technical advice and Dr. Xiaochun Zhao at PBI Cobbitty for assistance in growing rice plants. We also thank Dr. Jane Radford, Department of Pathology, University of Sydney for assistance with paraffin embedding.
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Received 7 December 2005/3 February 2006; accepted 9 March 2006doi:10.1016/j.cellbi.2006.03.004