Proteomic analysis revealed several differentially accumulated proteins (DAPs) in female (XX) and hermaphrodite (XYh) flower buds of contrasting genotypes at early and late stages of development. The time effect had a much greater influence on the protein profile than the sex effect, which indicates that the sex trait was attributed to a smaller set of genes.
The observed variations in protein accumulation among the papaya genotypes suggest that differential molecular processes underlie sexual differentiation. Notably, the greater number of DAPs in the SS72/12 genotype, particularly at the early stage of development, may indicate earlier gene activation related to sex determination in these plants. To focus on proteins potentially important for sexual differentiation across genotypes and developmental stages, the study specifically targeted those proteins with consistent accumulation patterns irrespective of genotype and time. This strategic approach allowed us to discern genotype-independent protein dynamics, facilitating the elucidation of core molecular processes driving flower development and differentiation.
The amount of DAPs was much greater in the SS72/12 genotype than in the other genotypes at both the early and late stages of development. The size of the flower buds was the standard used for sampling, which may have influenced the difference in the proteomic pattern, as both the development and the size of buds are different for each genotype. These results may indicate that gene activation was related to the sex of the SS72/12 plants earlier than to the other plants, as the other genotypes, mainly under early conditions, presented two times fewer DAPs between the sexes. This study focused only on proteins that exhibited the same accumulation pattern in the three studied genotypes due to differences in flower buds and with the aim of identifying proteins that play important roles in sexual differentiation regardless of genotype and time (Table 2).
Although transcriptome analyses focusing on the sexual differentiation of papaya have revealed several genes related to the biosynthesis of plant hormones and ABA, ROS, and auxin signaling pathways, most of the DAPs observed between the sexes in the present study were involved in carbohydrate metabolism, phenylpropanoid biosynthesis, and sporopollenin biosynthesis13,32,33. Furthermore, the genes encoding these proteins were all mapped to autosomal chromosomes or the autosomal region of chromosome 1. These discrepancies between transcriptomics and proteomics outcomes suggest that differences in post-transcriptional and post-translational regulation mechanisms could account for why certain genes identified in transcriptomics analyses may not correspond directly to the proteins identified in proteomics analyses. Additionally, variations in sample preparation, experimental conditions, and sensitivity of detection methods between transcriptomics and proteomics analyses could also contribute to differences in outcomes. In proteomics analyses, transcription factors are often not detected due to limitations in sensitivity detection compared to transcriptomics.
Differences in carbohydrate metabolism between female and hermaphrodite flower buds in Carica papaya
A large group of DAPs in papaya flower buds were related to metabolism. Most of the DAPs were involved in carbohydrate and amino acid metabolism and were up-regulated at the early and late stages of hermaphrodite bud development. These findings reinforce previous studies indicating that energy metabolism plays a crucial role in sexual differentiation and flower bud development34,35,36.
Studies of dioecious plants have shown differences in the energy expenditure between male and female individuals. Male flowers of Salix paraplexia and Silene latifolia have greater reproductive costs due to greater biomass accumulation and energy consumption during reproduction37. In the present study, compared with female flowers, hermaphrodite flower buds at the early stage of development presented an increase in the accumulation of proteins related to carbohydrate metabolism. Proteins involved in monosaccharide and amino acid metabolism were up-regulated at the final stage of development.
Many of the cited pathways are not directly related to specific structures or functions of the androecium or gynoecium, as the proteins involved in these pathways are expressed in both sexes but at different levels. Thus, they can be classified as secondary sexual characteristics, which arose to increase sexual specialization after the evolution of dioecy and trioecy38.
In the present study, the ATP-citrate lyase A-3 (ACLA-3) and acetyl-CoA carboxylase 1 (ACC1) proteins, which play important roles in energy metabolism, were up-regulated in hermaphrodite flower buds at an early stage of development. The first protein is involved in the generation of acetyl-CoA, and the second protein is involved in the carboxylation of acetyl-CoA in plants; these two proteins are important steps in the synthesis of carbohydrates, amino acids, fatty acids, and flavonoids39,40.
The protein 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), which was also up-regulated, interacted with the ACLA-3 and ACC1 proteins. HMGS is involved in the biosynthesis of phytosteroids, specifically the condensation of acetoacetyl-CoA and acetyl-CoA in the mevalonate (MVA) pathway. Informants with a mutation in the HMGS gene had infertile pollen grains, revealing that the tapetum requires the MVA pathway to develop tapetosomes and elaioplasts, which are required to form pollen coats41.
Most proteins enriched in carbohydrate metabolism in the present study play an important role in cell wall formation. Complex and highly dynamic plant cell walls are composed of interaction networks of polysaccharides, highly glycosylated proteins, and other polymers. This structure responds and adapts to normal processes of growth and development, as well as to biotic and abiotic stresses. Cellulose, hemicellulose, and pectins are the main carbohydrates of primary cell walls42.
The UDP-D-xylose synthetase (AXS2) protein and two probable proteins [pectin esterase/pectin esterase inhibitor (AT5G09760) and polygalacturonase (AT4G23500)] were up-regulated in papaya hermaphrodite flower buds at an early stage of development. The first two proteins are involved in synthesis43,44, and the latter is involved in pectin degradation45. Studies have shown that pectin is an essential factor for pollen development because it is the main constituent of primary cell walls46,47. Specifically, pectin is involved in the formation of the pollen grain wall, in addition to being a significant component of pollen carbohydrate reserves48.
UDP-D-glucose 4-epimerase (UGE5) is another protein identified in this work that is involved in pectin synthesis. This protein is involved in the synthesis of UDP-D-galactose, a nucleotide sugar precursor of several components of the cell wall matrix in addition to pectin, especially arabinogalactans, hemicellulose, and other polysaccharides and glycoproteins49,50.
Several extracellular glycosidic hydrolases play important but poorly understood roles in cell wall remodeling during plant growth51. In the present study, the enzyme xyloglucan endotransglucosylase/hydrolase 2 (XTH16) was up-regulated in papaya hermaphrodite flower buds at the early stage of development. The function of this enzyme is to promote cell wall plasticity through cleavage and reconnection of xyloglucan molecules, the main monosaccharide that makes up hemicellulose52. At least 33 genes have been reported in Arabidopsis to encode these enzymes53; the XTH3 homolog, expressed predominantly in Arabidopsis flower buds, seems to play a role in tapetum cell wall degradation52. Bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase (ASD1 and AT3G19620) is another glycosidic hydrolase that was DAP in hermaphrodite flower buds but was identified in buds at the final stage of development. This enzyme has already been identified in the roots and flowers of alfalfa (Medicago sativa L.) and has been characterized by the release of xylose and arabinose from the cell walls of these organs51.
The proteins glycosyl hydrolase 9B18 and 9B15 (GH9B18 and GH9B15, respectively) were unique to hermaphrodite flower buds at the early and late stages of development, respectively. Glycoside hydrolase family 9 (GH9) encodes a cellulase gene consisting of endo-β-1,4-glucanase, which is responsible for the process of cellulose synthesis and hydrolysis. Studies on the GH9 family of genes in plants have demonstrated their involvement in many plant development processes, such as cell elongation, anther dehiscence, pollen tube growth, abscission of branching nodes, and fruit ripening. More recently, GH9 genes characterized in wheat were shown to play important roles in anther development by targeting miRNAs and regulating cellulose levels via light and phytohormones54.
Proteins involved in pollen development in Carica papaya
The outer wall of pollen and spores, called the exine, is highly resistant to chemical reagents and enzymes. The exine not only provides a protective barrier against pathogen attack, dehydration, and ultraviolet (UV) irradiation but also facilitates pollen recognition and stigma attachment51. Extensive evidence suggests that exine formation is a highly conserved polyketide biosynthetic pathway present in all land plant lineages and is likely associated with plant evolution. The tetracetide alpha-pyrone reductase 1 (TKPR1), acyl-CoA synthetase (ACOS5), and hydroxyalkyl alpha-pyrone synthase (LAP6) proteins were unique to papaya hermaphrodite flower buds; the first was identified in early-stage flower buds, and the last two were identified in the final stage of development. These enzymes have already been well characterized in several species, such as A. thaliana, rice, tobacco, and rapeseed, and are involved in the biosynthesis of sporopollenin, the main component of the exine55,56.
The composition of sporopollenin is not fully understood. Additionally, the exine is considered one of the most complex cell walls in plants. However, recent studies have shown that derivatives of the phenylpropanoid pathway are essential components of sporopollenin and act mainly in UV protection and maintenance of the genomic integrity of pollen57. The enrichment of pathways related to sporopollenin and phenylpropanoid biosynthesis in hermaphrodite samples across both developmental stages reaffirms their pivotal role in papaya flower development. Furthermore, the predicted interactions between proteins involved in these pathways suggest coordinated regulation and functional integration, highlighting their collective contribution to the synthesis and assembly of sporopollenin and exine components. This finding reinforces the notion that metabolic pathways associated with sporopollenin and phenylpropanoid biosynthesis are indispensable for ensuring the proper development and reproductive success of papaya flowers.
The phenylpropanoid pathway also provides intermediates for the synthesis of lignin, flavonoids, and hydroxycinnamoyl esters58. Furthermore, studies have shown that the fluorescence profiles of xylem lignin and pollen wall exines are similar. The protein caffeic acid 3-O-methyltransferase (OMT1), a probable cinnamyl alcohol dehydrogenase 9 (CAD9), and another probable cinnamyl alcohol dehydrogenase 1 (CAD5), which are already well characterized as having a key role in the synthesis of lignin, were up-regulated in the flower buds of hermaphrodites, the first two at the early stage and the latter at the late developmental stage. Plant O-methyltransferases (OMTs) constitute a large family of enzymes involved in the methylation of the oxygen atom of several secondary metabolites, including phenylpropanoids, flavonoids, and alkaloids59. The CAD5 protein plays a key role in the lignification of the anther endothelium. Moreover, plants harboring this gene exhibit anther dehiscence and pollen release failure, which causes male sterility60. Like the homologs mentioned above, CAD9 has been detected in the stems, leaves, and flowers of Arabidopsis plants; however, its biochemical functions are unknown, and CAD9 is highly expressed in male flower organs61,62.
Class III peroxidases (PRXs) are glycoproteins that play important roles in cell wall maturation and lignin formation. The following four PRXs were up-regulated in the present study: peroxidase 17 (AT2G22420), peroxidase 40 (AT4G16270), putative peroxidase 48 (AT4G33870), and peroxidase 72 (AT5G66390). In Arabidopsis, the PRX72 gene is expressed in stems, roots, leaves, and flowers, while the PRX17 gene is expressed in stems, flowers, and silica. The two corresponding proteins are located in the cell wall and are involved in lignin accumulation63,64,65. In the present study, PRX72 was up-regulated in hermaphrodite buds at the initial stage but only at the final stage of development, whereas PRX17 was up-regulated only at the final stage of development.
PRX40, which accumulates at an early stage of development, plays an important role in tapetum development and, consequently, a key role in male fertility. Because the tapetum is not considered lignified or suberized, extensins stand out as a potential substrate for PRX4066. Extensins are structural glycoproteins known to regulate cell size and shape67. Finally, putative peroxidase 48 was unique to hermaphrodite samples at an early stage of development. This protein is also expressed in the mature stems of Arabidopsis68. Despite the lack of evidence about its real function, this PRX was identified for the first time in floral organs in the present study.
Although the present study did not identify proteins linked to sex determination in papaya, the results contribute to the understanding of floral development in this crop. Most of the up-regulated proteins in hermaphrodite flower buds seem to be related to the development of male flower structures, especially during pollen wall formation. Importantly, protein genes involved in lignin synthesis, similar to those identified here, were highly expressed in hermaphrodite flowers compared to male flowers of Lilium apertum69.
Among the three sexes in which papaya plants are present, hermaphrodite plants are more vulnerable to abnormal flower production, while female plants are more stable in terms of flowering. The deformation of hermaphrodite flowers may represent an evolutionary strategy for overcoming environmental stress. The theory that carpelloid and pentandric flowers represent a trend of hermaphrodite plants returning to their female form has already been proposed70. The present study suggested that hermaphrodite flowers require greater energy expenditure during pollen development, specifically for the formation of their complex outer wall.
This study used for the first time the shotgun comparative proteomics method to analyze proteins from female and hermaphrodite flower buds of C. papaya, providing information about important molecular events in floral development in papaya. The carbohydrate metabolism, phenylpropanoid biosynthesis, and sporopollenin biosynthesis pathways were more enriched in hermaphrodite buds than in female buds at both stages of development. Most of these proteins play key roles in pectin, cellulose, and lignin synthesis. These compounds play important roles in cell wall formation and seem to be related to the development of male structures in flowers, especially in the formation of the pollen coat. The results suggest that hermaphrodite flowers require greater energy expenditure during development, probably due to the formation of complex external pollen walls.
