In this work, the developmental differences between the PGMS MT and WT anthers were compared by cytological and proteomic analyses. Delayed tapetum degradation was confirmed in MT anthers at the UNP stage. To acquire information on the molecular mechanisms causing these developmental differences, we further analyzed the proteomes of MT and WT anthers at the TTP, UNP and BNP stages. Sixty-two differently expressed protein spots (represent 56 distinct proteins) were successfully identified. Based on their annotated biological and cellular functions, the 56 differentially expressed proteins could participate in a range of processes during pollen development, including energy and metabolic pathways, pollen wall development, protein metabolic, pollen tube growth, and other functional proteins. These results may help us to clarify the mechanism of male sterility in PGMS mutant.
Delayed degeneration of the tapetum in MT anthers
Formation of the anther is initiated by periclinal divisions in the hypodermal cells in the anther primordium. After mitotic divisions, the final structure consists of gametophytes surrounded by a series of cell layers, which are the tapetum, middle cell layer, endothecium and outer epidermis . These layers, especially the tapetum, play important roles in pollen development, such as the production of the locular fluid and callase, and the formation of exine precursors . Tapetal degeneration is induced through PCD during the late developmental stage of the anther, and premature or delayed degradation causes male sterility ,.
In our study, the tapetum of the WT anther started to degenerate at the early UNP stage (Figure 2C), and little remained in the locule at the BNP stage (Figure 2D). However, the tapetum failed to degenerate in the MT anthers at an appropriate stage (Figure 2I), and most still remained at late UNP stage (Figure 2J). Because of the delayed tapetum degradation in the MT anthers, no enough nutrients were available for normal microspore development. As a consequence, the MT pollen underwent abnormal development, resulting in male sterility.
Arabidopsishomologues affected in pollen development
To find out how cotton pollen might differ from Arabidopsis pollen, we compared the proteins in our study to the proteins identified in pollen proteome of Arabidopsis -. Fifty of the 56 identified proteins could be assigned to an Arabidopsis homologue, indicating high similarity of the proteomes. The difference may result from the different samples used. In this study, the whole anther was sampled for protein extraction and identification, not the separated pollen grains as the Arabidopsis proteome. Chalcone synthase (CHS, spot 137) was significantly down-regulated in MT anther in all three stages (Table 1). Its Arabidopsis homologue AT4G34850 (LAP5 and LAP6) was absent in pollen proteome. LAP5 and LAP6 are male-organ-specific members and are expressed in anthers coincident with the timing of exine formation .
In Arabidopsis, many mutants are described that are affected in pollen development and pollen tube growth. A total of 215 genes have been surveyed by Till Ischebeck et al. . From these, we found five spots to have homologues (E-value equal to or less than 10−10) in our study. Although the roles of these genes have not been discussed in cotton anther, the high similarity suggested conserved functions. Their changed expression pattern led to aberrant pollen development in MT anther, resulting in male sterility. However, from the 56 proteins identified, only 5 homologues have been described so far in Arabidopsis mutant studies, leaving tremendous room for future pollen research.
Energy and metabolism processes
In higher plants, the development of the male gametophyte is a well-programmed and elaborate process , which may require more genes expression. For example, compared with other organs, more than 20,000 genes have been detected as expressing in cotton anthers . To accomplish this complex process, numerous proteins are associated with energy and metabolism in anther development. It has been well studied in Arabidopsis thaliana. For example, of the proteins identified on the 2-DE reference proteome maps for mature pollen of Arabidopsis thaliana, ~40% are predicted to function in metabolism and energy generation ,. Except for these 2-DE based proteomics analysis, the metabolism and energy functional categories were also overrepresented in a shotgun proteomics of Arabidopsis pollen . Moreover, the tobacco proteome analysis from early to late pollen development demonstrated that proteins involved in primary metabolism and starch synthesis, which were required for pollen tube growth . These suggested that energy and metabolism processes were the most primary processes in pollen. Disordered expression of proteins in these processes may cause male sterility ,. In this study, ~31% (19) of the 62 spots identified were implicated in energy and metabolism (Table 1). Their up- or down-regulation may cause abnormal development of the MT anthers.
In detail, two of the proteins identified here had functions in carbohydrate metabolism (spot 38 representing triosephosphate isomerase, and spots 164 and 165 representing galactose oxidase), two in energy generation (spot 18 representing ATP synthase, and spots 48 and 99 representing NADH dehydrogenase) and the others were involved in metabolism processes. As the highest sink, anthers need to obtain large amounts of sugars to support their early development, and at later stages pollen maturation requires the accumulation of starch, which functions as an energy reserve for germination, thus serving as a marker of pollen maturity . It has been shown that changes in expression of carbon and energy metabolism genes led to total soluble sugar content decrease at the meiosis and UNP stages in the cotton GMS mutant anthers . In this study, because of the altered expression levels of carbohydrate metabolism-related genes, there was a strikingly reduced accumulation of sugars in the MT anthers at the late developmental stages (Figure 3A) and limited starch synthesis in the MT mature pollen grains (Figure 3C). As expected, the WT mature pollen grains (Figure 2F) stored a number of substances (e.g., polysaccharides, proteins, lipids and hormones) that place a high demand on energy and carbon reserves for successful germination and tube growth . But the MT pollen grains were nearly empty from late UNP stage (Figure 2J). During anther development, there is an increased demand for respiratory function and cellular energy in the form of ATP. Defective in ATP synthesis may result in abnormal anther development with non-functional pollens ,. In this work, two proteins in energy generation (spot 18 representing ATP synthase, and spots 48 and 99 representing NADH dehydrogenase) were significantly reduced in MT anthers, suggesting that the MT anthers were in an energy starved state.
These results suggest that the disordered gene expression in carbohydrate metabolism and energy germination resulted in reduced accumulation of total sugars, a lack of starch and other substances synthesis in the MT pollen grains, thus providing critical information augmenting our understanding of male sterility.
Pollen wall development
The pollen wall is formed of a number of layers, the outer exine, the outer sculptured layer or sexine and the inner nexine. The exine layer is formed principally of sporopollenin, which is synthesized predominantly by the tapetum and is an aliphatic polymer comprised of a series of polymers derived from long-chain fatty acids, phenylpropanoids and oxygenated aromatic rings . Its primary roles are to provide structural and physical support to the microspore cytoplasm and protection from harsh conditions, such as prolonged desiccation, high temperatures and ultraviolet light. It also facilitates pollination by attracting vectors that prefer an elaborate pollen outer wall . In Arabidopsis, defects in sporopollenin formation can cause male sterility . In this study, four proteins, represented by spots 137, 140, 141 and 145, that are involved in sporopollenin formation were found to be differentially expressed in the MT anthers (Figure 4).
Chalcone synthase (CHS, spot 137), which is the first committed enzyme in the biosynthesis of all flavonoids and is essential for pollen development and the biosynthesis of sporopollenin , was significantly reduced in all three stages of MT anther development. In Arabidopsis, LAP5 and LAP6 encode anther-specific proteins with homology to CHS and may play a role in the synthesis of pollen fatty acids and phenolics found in exine. Mutations in either gene result in abnormal exine patterning, whereas the lap5 lap6 double mutant produces pollen grains devoid of exine, causing strong male sterility .
The proteins represented by spots 140, 141 and 145, which were classified into the fatty acid synthesis pathway, were significantly reduced as well in the MT. Fatty acids are important components of sporopollenin. Mutations that in fatty acid synthesis can cause impaired pollen wall formation . Enoyl-[acyl-carrier-protein] reductase (EACPR, spot 145) is a subunit of the fatty acid synthase complex that catalyzes de novo synthesis of fatty acids. A reduced-function mutation of this gene in Arabidopsis, mosaic death1 (mod1), causes a marked decrease in its enzymatic activity, impairing fatty acid biosynthesis and decreasing the amount of total lipids .
The pyruvate dehydrogenase E1 component subunit beta (PDH E1-B, spots 140 and 141) is essential for the synthesis of sporopollenin precursors. Acetyl-CoA is formed from pyruvate through a PDH complex in mitochondria, and the released acetyl-CoA is a substrate for de novo fatty acid synthesis in plastids . Antisense inhibition of PDH_E1α-1 in the anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS . The relatively reduced amount of sporopollenin formation–related proteins in the MT anthers could contribute to male sterility. Because fatty acids are the likely components of sporopollenin, which contributes to the formation of the protective pollen coat , reduced amounts of these proteins may lead to abnormal pollen coat formation in the MT. The MT pollen was irregularly shaped (Figure 1K), which may have resulted from abnormal sporopollenin formation. To uncover the detailed changes, the structure of the pollen wall will be further studied at high resolution microscope.
As a non-photosynthetic male reproductive organ, the anther needs to obtain nutrients from source organs to support pollen development and maturation, and proteins, as well as amino acids, are important components of pollen cytoplasm ,. Proteasomes are important proteases in eukaryotes and regulate many cellular processes, including metabolism, cell cycle and the proteolysis of regulatory proteins. The altered expression levels of proteasome-related enzymes in the tomato 7B-1 anthers may affect meiosis in the microspore mother cells . In our previous research , several genes related to the ubiquitin-proteasome system were up-regulated in the MT anther at the UNP stage. Thus, under long-day conditions, the ubiquitin-proteasome system is induced in the MT at the UNP stage and likely leads to protein degradation. With insufficient protein and amino acid levels, the cytoplasm of MT pollen grains is likely to break down gradually, and the pollen grains are likely to lose activity, resulting in male sterility .
In this study, the proteolytic enzymes proteasome subunit α type-2-B (spot 39), proteasome subunit beta type-1 (spot 168) and 26S protease subunit 6B homolog (spot 63) (Figure 4), were up-regulated in the MT and one amino acid biosynthesis–related protein, 3-isopropylmalate dehydratase small subunit (spots 37 and 155), was down-regulated in the MT anthers. These changes may cause reduced protein and amino acid levels in MT pollen grains, although the exact mechanism for this effect is still unclear. Consistent with the previous study, the induced degradation of cytoplasmic proteins in the pollen of the MT may be another reason for its male sterility.
Pollen tube growth
Pollen germination, along with pollen tube growth, is an essential process in the reproduction of flowering plants. The wall of the pollen tube is composed of a single layer of pectin, and pectin methylesterases (PMEs) likely play a central role in pollen tube growth and the determination of pollen tube morphology . The function of PMEs in pollen tube growth and pollen germination has been well studied in several plant species. AtPPME1 is a pollen-specific gene, and its protein is found only in the mature pollen grains and growing pollen tubes. After germinating and being cultured in vitro, pollen tubes of atppme1 mutant pollen grains have a curved, irregular morphology and are dramatically stunted .
In plants, PME activities are regulated by either differential expression or posttranslational modification by specific PME inhibitor proteins (PMEIs) . It has been suggested that AtPMEI2 accumulates exclusively at the pollen tube apex and regulates pollen tube wall stability by locally inhibiting PME activity . Additionally, the ectopic expression of a BoPMEI1 antisense gene in Arabidopsis suppresses expression of its orthologous gene, At1g10770, resulting in pollen tube growth retardation, partial male sterility, and reduced seed set .
LAT52 is also essential for pollen development, because pollen grains that express antisense LAT52 RNA hydrate and germinate abnormally and cannot achieve fertilization . Interestingly, all the three related proteins in this analysis (represented by spots 166 and 173 for the PME; spots 103, 138 and 143 for the PMEI and spots 102 and 122 for LAT52) had lower expression levels in our MT anther maps and extremely high expression levels in the WT maps, especially at the BNP stage (Figure 4). We believe that this change may reduce the accumulation of pollen components for pollen tube growth, which leads to pollen that could not germinate after maturation, resulting in nonviable pollen grains.
Other functional proteins
The 27 remaining proteins could be classified into other diverse functional categories (Table 1). They have important roles in anther development as well, including five proteins with roles related to protein folding and assembly. The 23.6-kDa heat shock protein (HSP; spot 20), 17.3-kDa HSP (spot 91), protein disulfide-isomerase (spot 59) and elongation factor Tu (spot 71) were up-regulated in MT anthers; however, HSP70 (spot 124) was down-regulated. These proteins have been well studied and are responsible for protein folding and assembly . In this study, the different expression levels of HSPs implied that protein folding and assembly are altered in MT anthers, suggesting variations in protein translation and post-translational modifications, which might lead to aberrant anther development.
Stress defense–related proteins formed another functional category that included L-ascorbate peroxidase (APX; spots 126, Figure 4) and aldehyde dehydrogenase (ALDH; spots 100, 170 and 171), which were down-regulated in the MT. These proteins are important in detoxifying reactive oxygen species damage during anther development in upland cotton . Thus, the differential expression of these proteins in MT anthers may unbalance the oxidation-reduction process, which may play an important role in anther and pollen development ,. In addition, other proteins with significantly altered expression levels may also influence anther and pollen development. Further studies are required to investigate the functions of these proteins.