Seed Coat Degradation and Viability Loss in Pomegranate: The Hidden Cause of Aril Paleness Disorder

Seed Coat Degradation and Viability Loss in Pomegranate: The Hidden Cause of Aril Paleness Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Seed Coat Degradation and Viability Loss in Pomegranate: The Hidden Cause of Aril Paleness Disorder Mehdi Rezaei, Parviz Heidari, Stefanie Reim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9112637/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Pomegranate aril paleness (AP), a physiological disorder linked to climate change, causes pale, desiccated arils in otherwise healthy fruits, reducing marketability. To investigate the anatomical and physiological underpinnings of AP, this study examined four pomegranate cultivars exhibiting varying degrees of susceptibility: the resistant cultivar 'Damavand' ( DN ), the moderately affected cultivars 'Kashmar' ( KN and KW ), and the severely affected cultivar 'Torud' ( TW ). Seed viability was assessed using germination tests and tetrazolium staining, while structural abnormalities in the seed coat were assessed through histological analysis and scanning electron microscopy (SEM). The results revealed a strong association between AP severity and reduced germination rates, with germination rates ranging from 45% in the resistant cultivar ‘DN’ to just 4% in the severely affected cultivar ‘ TW’ . Interestingly, tetrazolium tests indicated that a large proportion of embryos remained viable despite severe AP symptoms, ranging from 67% in ‘ TW’ to 98% in ‘DN’ . Microscopic analyses further demonstrated substantial structural degradation in the seed coats of AP-affected cultivars, including blackened regions, tissue separation, and reduced cellular density in the inner seed coat layers. Taken together, the findings highlight that AP is associated with specific anatomical abnormalities in the seed coat and tegmen, which may underlie or exacerbate the physiological manifestations of the disorder. Biological sciences/Physiology Biological sciences/Plant sciences Punica granatum seed viability embryo health scanning electron microscopy (SEM) physiological disorder Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The pomegranate ( Punica granatum ) is an economically important fruit cultivated in subtropical and temperate regions with mild winters (Yilmaz et al., 2021 ). Its high antioxidant content has generated substantial global interest, contributing to its growing market demand (Sarkhosh et al., 2021 ). Pomegranate shrubs exhibit notable tolerance to drought and nutrient-poor soils, making them well-suited to arid and semi-arid environments. Major pomegranate-producing countries in the world include Iran, Turkey, India, Spain, and the United States (Yilmaz et al., 2021 ). In Iran, most pomegranate orchards are located on the edges of desert regions characterized by mild winters (Ebrahimi, 2015 ). However, in recent decades, coinciding with climate change, the incidence of physiological disorders such as fruit cracking and sunburn has increased significantly. Among these, a newly recognized physiological disorder referred to as "Aril Paleness" (AP) or "Aril Browning" has emerged as a major concern in many pomegranate-growing regions of Iran (Tabar et al., 2009 ). AP-affected pomegranate fruits exhibit pale or browned arils with a dry, compromised internal texture, despite an outwardly healthy appearance. This not only reduces their appeal for fresh consumption but also makes them unsuitable for industrial processing, thereby impacting commercial value. Consumers often purchase pomegranates that appear healthy from the outsiden, only to find a low-quality product upon consumption. This has led to a decline in consumer trust and demand, creating significant challenges for quality control, particularly in export markets (Tabar et al., 2009 ). Although several studies have proposed practical approaches to mitigate this physiological (Kavand et al., 2017 ; Moradi et al., 2024 ; Tadayon, 2021 ), none have proven consistently effective in reducing this disorder. One consistent finding across these studies is the existence of cultivar-specific susceptibility, with some genotypes showing higher resistance to the disorder than others (Kavand et al., 2017 ; Shivashankar et al., 2012 ). In affected fruits, arils fail to accumulate anthocyanin pigments, resulting in pale coloration. Additionally, the aril texture becomes sugary, slightly dehydrated, and develops an undesirable taste. When more than 50% of the arils are affected, the fruit is considered unfit for consumption. Numerous researchers have cited elevated temperatures during fruit development, linked to climate change, as key factor for the lack of pigment deficiency in pomegranate arils (Kavand et al., 2017 ; Melgarejo et al., 2024 ; Tabar et al., 2009 ; Tadayon, 2021 ). However, the precise physiological and anatomical mechanism underlying this disorder remain poorly understood. Traditionally, the edible portion of the pomegranate fruit, the aril, has been described as a highly enlarged single cell. However, more recent anatomical studies by Melgarejo et al. ( 2024 ); Pujari and Rane ( 2015 ), suggest that the aril comprises the entire seed structure, including the testa (outer integument), tegmen (inner integument), and the embryo with its cotyledons. The testa is notably thick, consisting of 5 to 18 layer of cells, while the tegmen is narrower and typically composed of 4 to 6 cell layers (Pujari & Rane, 2015 ). Within the testa, two main components are distinguished: the fleshy, edible sarcotesta, and the thick-walled, hard, sclerotic mesotesta. During fruit ripening, cells in the testa undergo a considerable expansion, contributing to the development of the pulpy sarcotesta (Melgarejo et al., 2024 ). This tissue acts as a reservoir for water and bioactive compounds, contributing to the fruit’s sensory and nutritional qualities (Pujari & Rane, 2015 ). The tegmen is also a multilayered structure, generally composed of an outer layer (4 to 5 cells thick) and an inner single-celled epidermis (Pujari & Rane, 2015 ). Melgarejo-Sánchez et al. ( 2021 ) demonstrated that changes in aril pigmentation correlates closely with fruit ripening stages, highlighting the importance of integument development in fruit quality. Based on these insights, our study investigates the anatomical abnormalities and viability of arils in pomegranate cultivars exhibiting varying degrees of aril paleness disorder (AP) different levels of disorder. We aimed to elucidate the underlying mechanisms of AP by assessing seed vigor, embryo viability, and the structure integrity of the surrounding integument tissues. Materials and Methods Plant Materials Pomegranate fruit samples were collected at commercial harvesting maturity from orchards in two distinct regions of Shahroud County, Iran: Mayami (36.2433° N, 55.3910° E) and Torud (36.4259° N, 55.0139° E). These sites were selected to represent two climatically different regions in Iran (Fig. 1 ). The samples were categorized according to the presence and severity of AP: ‘DN’ ( 'Damavand' cultivar with normal arils, exhibiting no paleness), ‘KN’ ( 'Kashmar' cultivar with normal arils, exhibiting slight paleness), ‘ KW’ ( 'Kashmar' cultivar exhibiting prominent AP), and ‘ TW’ ( 'Torud' cultivar exhibiting severe AP) (Fig. 2 ). ‘DN’ , ‘ KW’ and 'KN' samples were sourced from the Mayami region of Shahroud County in Iran, an area with a relatively mild climate. The 'TW' samples originated from the Torud region in Iran known for its warmer climate (Fig. 1 ). Fruits were harvested at full maturity, based on visual assessment of skin color and fruit size. From each cultivar, 12–25 fruits were randomly collected from three trees within commercial orchard. Seed Viablity Tests Pomegranate seeds were extracted from three randomly selected fruits per cultivar. Following extraction, the seeds were cleaned with water to remove any remaining pulp, after which they were air-dried at an ambient temperature of approximately 25°C for seven days. The seeds were surface-sterilized by immersing them in a 1% sodium hypochlorite (NaOCl) solution for 10 minutes, followed by three rinses with sterile distilled water. A gibberellic acid (GA 3 ) treatment was also employed to promote germination. The seeds were imbibed by being soaked in either a 500 mg/L GA 3 solution or sterile distilled water (control) for 12 hours under dark conditions. Following imbibition, groups of 25 seeds were transferred to sterile Petri dishes (90 mm diameter) containing two layers of Whatman® No. 1 filter paper that had been moistened with sterile distilled water. The experimental design was completely randomized, with four replicates per treatment. Petri dishes were sealed with Parafilm to minimize moisture loss and incubated in a controlled-environment germination chamber maintained at a constant temperature of 25 ± 1°C with a photoperiod of 16 hours. Germination was monitored daily for a duration of 50 days, starting from the third week after sowing. A seed was considered germinated when the radicle emerged by at least 2 mm. Germination parameters were calculated as follows: Germination percentage (GP) = (Ng / Nt) × 100, where Ng is the number of germinated seeds and Nt is the total number of seeds tested. Germination vigor (GV) = Σ (germinated seeds on day n × n) / total seeds. (higher values indicate greater vigor). A tetrazolium test was also conducted to assess the viability of embryo and cotyledon tissues. The seeds were initially pre-conditioned by soaking them in sterile distilled water for 24 hours to soften the tissue. Following imbibition, the pomegranate seeds were carefully dissected using a sterile scalpel under aseptic conditions. The embryos and cotyledons were then excised from the seeds in an aqueous environment using a fine needle to minimize mechanical damage. Excised tissues were then immersed in a 0.1% (w/v) tetrazolium chloride (2,3,5-triphenyltetrazolium chloride; TTC) solution prepared in phosphate buffer (50 mM, pH 7.0) and incubated in the dark at 25°C for 12 hours. During incubation, viable tissues reduce the colorless TTC to red-colored formazan. Following incubation, tissues were rinsed three times with distilled water to remove excess TTC. The extent and intensity of formazan staining in the embryo and cotyledon tissues were visually assessed and scored under a stereomicroscope (Olympus SZX10). Thirty seeds from three separate fruits were evaluated per treatment. Histological analysis -Seed Coat Abnormality To examine the internal structure of the seed coat, fresh pomegranate seeds were extracted in an aqueous solution to prevent desiccation. The seeds were bisected with a sterile scalpel and the embryo and cotyledons were carefully removed. The internal surface of the seed coat was then examined and photographed under a stereomicroscope (Olympus SZX10) to assess its cellular structure and identify any abnormalities. A total of thirty seeds from three separate fruits were evaluated, with three fruits taken from each pomegranate cultivar. - Scanning Electron Microscopy and Energy-Dispersive Spectra Based on stereomicroscope observations of affected tissues within the hard seed coat, the tegmen and the mesotesta, samples of the hard seed coat were prepared for scanning electron microscopy (SEM). The samples were initially dried in a desiccator containing silica gel until they reached a constant weight to ensure complete dehydration. The dried samples were then mounted on aluminium stubs using carbon tape and sputter-coated with a thin layer of gold (approximately 20 nm) in a vacuum using a tabletop sputter coater (Cressington 108auto) to enhance conductivity and prevent charging during imaging. Images were acquired at various magnifications (ranging from 500x to 2000x) using a field emission scanning electron microscope (FESEM, Zeiss Sigma 300-HV, Germany) operated at an accelerating voltage of 5 kV. In addition, elemental analysis was performed on the surface of each sample using energy-dispersive X-ray spectroscopy (EDS, Oxford Instruments) which was integrated with the FESEM. Three samples per cultivar were analyzed, and EDS spectra were collected from multiple points on each sample to ensure representative elemental composition data. Results -Seed Viability Tests The germination study revealed clear disparities in germination potential among the pomegranate cultivars. A striking correlation was observed between AP and reduced germination percentage and rates (Fig. 3 ). The 'DN' cultivar, which exhibited no AP, demonstrated the highest germination percentage, highlighting its robust seed viability. Conversely, the 'TW' and 'KW' cultivars with pronounced AP, showed markedly reduced germination, indicating a detrimental effect of the paleness on seed quality. While the 'KN' cultivar with slight paleness displayed intermediate germination, it was still significantly lower than that of the 'DN' cultivar (Fig. 3 ). Interestingly, the application of gibberellic acid (GA 3 ) did not consistently improve germination rates across all cultivars. In fact, a slight reduction in germination rates was observed in the 'DN' cultivar under GA 3 treatment, though the high variability of the results makes this inconclusive. As can been seen in Fig. 3 , the 'DN' cultivar exhibits the highest germination rate under both control and GA3-treated conditions. The control group of 'DN' has a germination rate of approximately 17 seeds per week, while the GA3-treated group shows a rate of around 13 seeds per week. The ‘KN’ cultivar , exhibits a lower germination rate compared to the 'DN' cultivar. The ‘KW’ and 'TW' cultivars exhibit even lower germination rates than ‘ KN’ cultivar. Despite the significant differences observed in germination tests among cultivars with varying degrees of AP disorder, the tetrazolium viability tests of embryo revealed that the examined pomegranate cultivar with extreme AP, 'TW' , still maintained over 67% healthy and viable embryonic tissue (Fg. 3, Fig. 4 A). However, a statistically significant difference was found between cultivars exhibiting AP and those with normal arils; the 'KN' and 'DN' cultivars showed over 98% viable embryos (Fig. 3 ). -Histological analysis A histological examination was conducted on seeds from each cultivar using longitudinal sections to observe the internal structures (Fig. 4 and Fig. 5 ). In the ‘ TW’ cultivar, over 90% of the seeds exhibited a distinct blacking region at the distal end of the inner integument (tegmen) (Fig. 4 B, C, D), whereas in ‘ DN’ no blacking of the tegmen was observed (Fig. 4 , Ci and Di). Around 20% of these seeds showed tissue degradation (Fig. 5 B, C), and in a few arils, the embryo was not formed (Fig. 5 A). This blackening in tegmen was also visible in 58% of the seeds from the ‘ KW’ cultivar, whereas only 10% of ‘DN’ and ‘ KN’ seeds showed this discoloration in the inner tegmen. Analysis of SEM images of seed coats from different pomegranate cultivars revealed a clear relationship between AP and the structural integrity of the seed coat (Fig. 6 ). The 'DN' cultivar, which exhibited no AP, showed a healthy and intact seed coat structure, characterized by well-organized sclereid cells and a distinct interface with the inner integument (Fig. 6 , picture DN ). In contrast, cultivars with AP (‘ KW’ and ‘ TW’ ) displayed marked degradation and disruption of the seed coat, particularly in the sclereid cell layer and at the interface with the inner integument. These changes were evident as collapsed cells and structural disorganization (Fig. 6 , pictures KW and TW ). The 'KN' cultivar, showing mild AP symptoms, exhibited an intermediate level of degradation (Fig. 6 , picture KN ). These results suggest that AP may be associated with processes that compromise the development or maintenance of seed coat structure, potentially reducing seed viability. Supporting this, Fig. 7 shows that seed coat cells in healthy cultivars had thicker, more intact cell walls compared to those affected by AP. DN Figure 7. Scanning electron microscope (SEM) images of inner sclereid cells of mesotesta in the seed coat of pomegranate cultivars with and without AP symptoms. The affected samples show thinner cell walls and smaller cell size, indicating structural degradation associated with aril paleness. The middle image and highlighted area show the selected region of the seed coat used for the SEM image. ‘DN’ : 'Damavand' cultivar without aril paleness, ‘ KN’ : 'Kashmar' cultivar without aril paleness, ‘ KW’ : 'Kashmar' cultivar with aril paleness, ‘ TW’ : 'Torud' cultivar with aril paleness. -EDS analysis Comparative EDS (Energy Dispersive X-ray Spectroscopy) analysis of seed coats from different pomegranate cultivars revealed distinct elemental differences associated with varying levels of AP susceptibility (Table 1 ). In this study, elemental variations observed between cultivars provide further evidence of physiological differences related to seed coat integrity and the occurrence of AP. The highly susceptible cultivar 'TW' showed a 5.6% reduction in oxygen concentration (41.72 wt%) compared to the resistant cultivar 'DN' (44.05 wt%), along with a corresponding increase in carbon levels (‘ TW’ : 57.99 wt% vs. ‘ DN’ : 55.23 wt%). Calcium levels were remarkably lower in ‘TW’ (0.05 wt%) than in ‘DN’ (0.16 wt%), reflecting a 68.7% decrease. Potassium content was also reduces in ‘ TW’ (0.17 wt%) compared to ‘ DN’ (0.32 wt%), representing a 47% decline. Interestingly, sulfur was uniquely detected in the moderately affected 'KW' cultivar (0.10 wt%), possibly indicating stress-induced accumulation of sulfur-containing metabolites. Magnesium was exclusively present in ‘ TW’ (0.06 wt%), suggesting atypical cationic substitution within lignified strata. Together, these elemental deviations point to cultivar-specific differences in seed coat composition and integrity, elucidating the physiological mechanisms of impaired nutrient transport and weakened cell adhesion in AP-susceptible tissues. Table 1 Comparative elemental composition of seed coats across pomegranate cultivars with varying AP susceptibility Element ‘DN’ )wt%( ‘ KN’ (wt%( ‘ KW’ (wt%) ‘ TW’ (wt%) C 55.23 ± 0.64 53.84 ± 1.61 56.98 ± 0.35 57.99 ± 0.41 O 44.05 ± 0.63 46.16 ± 1.61 42.77 ± 0.35 41.72 ± 0.41 Ca 0.16 ± 0.06 ND ND 0.05 ± 0.03 K 0.32 ± 0.06 ND 0.15 ± 0.02 0.17 ± 0.03 Mg ND ND ND 0.06 ± 0.03 S ND ND 0.10 ± 0.02 ND Cl 0.24 ± 0.05 ND ND ND ND = Not Detected (± 0.01 wt% detection limit). ‘DN’ : 'Damavand' cultivar without paleness, KN: 'Kashmar' cultivar without paleness, KW: 'Kashmar' cultivar with paleness, TW: 'Torud' cultivar with paleness Discussion This study investigated anatomical and physiological traits linked to aril paleness disorder (AP) in four pomegranate cultivars with varying susceptibility. Seed viability and seed coat structure were analyzed to identify factors that may explain differences in AP resistance. Our findings provide robust evidence that AP disorder in pomegranates is primarily driven by critical impairments in embryo development, compounded by structural deficiencies in the seed coat. Embryo Viability as the Primary Driver of Aril Paleness Disorder (AP) The viability tests revealed that aril paleness disorder (AP) in pomegranates is strongly associated with a drastic reduction in seed germination capacity and a high incidence of non-viable embryos in affected fruits. In particular, the highly susceptible cultivar ‘Torud’ (TW) exhibited a germination rate of only 4%, compared to 45% in the resistant cultivar ‘Damavand’ (DN), despite both being cultivated under identical management practices. These findings are consistent with previous research by Shivashankar et al. ( 2012 ), who also identified the seed as the primary origin of aril paleness disorder. Several factors may contribute to impaired embryo development and reduced seed viability, including cultivar specific susceptibility or environmental stresses such as elevated temperatures and drought (Faraji and Karami, 2024 ). This is supported by the highly susceptible cultivar ‘ TW’ with most pronounced seed coat abnormalities, which is growing in the warmer regions like Toroud. In addition, nutrient imbalances and disruptions in phytohormone signaling, which is normally mediated by the embryo to regulate fruit development, may also play a role. Genetic predisposition appears to be a key factor as well, with resistant cultivars like ‘ DN’ likely possessing inherent mechanisms that support normal embryo development even under stress(Mohammad Kavand et al., 2017 ). The connection between impaired embryo development and aril degradation is multifaceted. A non-functional embryo loses its role as a metabolic sink, leading to the accumulation of sugars and other metabolites in the aril tissue. This metabolic imbalance disrupts anthocyanin biosynthesis, which is resulting in pale coloration, and increases oxidative stress, which in turn activates browning-related enzymes. Supporting this, Meena et al. (2021) reported that necrosis in the innermost mesocarp tissues during endocarp hardening coincides with the onset of aril browning. Altogether, the physiological collapse of the embryo and surrounding tissues initiates a cascade of metabolic and structural deterioration that culminates in the visible symptoms of AP(Shivashankar et al., 2012 ). Seed Coat Structure and Elemental Integrity as Key Modulators of AP Expression Furthermore, our study highlights severe degradation of sclereid cells and inner integument layers in affected AP pomegranates, evidenced by SEM images showing cell separation. This structural damage directly impedes solute transfer to developing arils and compromises the seed coat's protective function, making it vulnerable to stress. The structural integrity of the seed coat, particularly the sclerotic mesotesta, is vital for nutrient transport. Our EDS findings of calcium depletion in AP-susceptible cultivars (‘ TW’ : 0.05 wt% vs. ‘ DN’ : 0.16 wt%) further align with Pujari & Rane's (2015) work, which established calcium's role in the lignification and rigidity of mesotesta cells. This suggests that AP may involve climate-disrupted calcification of these sclerotic layers, impairing vascular transport to the arils. The coincidence of the 45-day post-anthesis sclerification window (Pujari & Rane, 2015 ) with our observed temperature sensitivity period is critical, strongly suggesting that AP mitigation strategies should target early fruit development through timely calcium-boron supplementation to ensure proper sclerification and overall seed coat health, thereby protecting embryo development and maintaining fruit quality. In addition, EDS (Energy Dispersive X-ray Spectroscopy) revealed an altered C:O ratio in ‘ TW’ (1.85) compared to ‘ DN’ (1.67), suggesting degradation of oxygen-abundant pectic polysaccharides in the cell wall matrix (Anderson, 2019), consistent with middle lamella disintegration observed in SEM imagery. This likely disrupts pectate cross-linking essential for cell wall cohesion (Wdowiak et al., 2024). As EDS is a well-established method for detecting nutritional and elemental changes in plant tissues (Kopittke et al., 2020; van Der Ent et al., 2018; Wyroba et al., 2015), these findings provide further evidence that cell wall integrity in AP-affected seed coats is severely compromised. Ultimately, both embryo viability and seed coat structure must be considered in a comprehensive understanding of AP, as both elements appear to interact and amplify the disorder under unfavorable environmental conditions. These findings suggest that AP mitigation should focus on early fruit development, particularly during the sclerification window. Targeted nutrient management, including calcium and boron supplementation, may support proper seed coat formation and protect embryo development, thereby improving overall fruit quality and resistance to AP. Cultivar selection also plays a crucial role, and breeding programs may benefit from prioritizing genetic lines with inherent embryo resilience and seed coat integrity. Conclusion In summary, this study demonstrates that aril paleness disorder in pomegranates is primarily associated with impaired embryo development and compromised seed coat integrity. Both factors interact synergistically, especially under environmental stress, to promote AP expression. However, the limited number of cultivars examined and the lack of multi-site environmental replication suggest caution in generalizing these findings. Future research should focus on controlled environmental studies and molecular investigations of embryo development to confirm and expand upon these insights. Early-stage nutrient interventions and cultivar selection nevertheless emerge as promising strategies to mitigate this economically significant disorder and improve overall fruit quality. Abbreviations AP Aril paleness DN 'Damavand' pomegranate cultivar with normal arils KN 'Kashmar' cultivar with normal arils exhibiting slight paleness KW 'Kashmar' cultivar exhibiting prominent AP TW 'Torud' cultivar exhibiting severe AP SEM Scanning electron microscopy GA3 Gibberellic acid GP Germination percentage GV Germination vigor TTC 2 3 5-triphenyltetrazolium chloride EDS Energy-dispersive X-ray spectroscopy. Declarations Ethics, Consent to Participate, and Consent to Publish declarations All plant materials used in this study were obtained with proper authorization. Pomegranate ( Punica granatum L.) samples were collected from three privately-owned commercial orchards in Mayami (36.24°N, 55.39°E) and Torud (36.62°N, 55.01°E), Shahroud, Iran, following agreements with the owners (Mr. Abedian and Mr. Mirii). Voucher specimens (POM-2025-001 to POM-2025-004) have been deposited in the Herbarium Lab., Agriculture Faculty, Sharood University of Technology, Shahrood, Iran. Conflict of Interest: The authors declare that they have no conflicts of interest. Clinical trial number not applicable Funding Declaration: This work was supported by the Center for International Scientific Studies & Collaboration (CISSC), Ministry of Science, Research and Technology of Iran. Author Contribution Authors' contributions: MR and PH conducted data collection and experiments. MR supervised the project and wrote the manuscript. MR, PH, and ER contributed to conceptualization and data interpretation. All authors reviewed and approved the final manuscript. Data Availability All study data are included in the manuscript. Additional datasets are available from the corresponding author upon reques References Faraji, S. & Karami, S. Spatial distribution of pomegranate aril paleness and its relationship with some environmental and non-environmental factors using geographic information system (GIS). Iran. J. Hortic. 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Aril Paleness, New Physiological Disorder in Pomegranate Fruit (Punica granatum): Physical and Chemical Changes during Exposure of Fruit Disorder: Physical and Chemical Changes during Exposure of Fruit Disorder. Hortic. Environ. Biotechnol. 50 (4), 300–307 (2009). Tadayon, M. S. Effect of foliar nutrition with calcium, boron, and potassium on amelioration of aril browning in pomegranate ( Punica granatum cv.‘Rabab’). J. Hortic. Sci. Biotechnol. 96 (3), 372–382 (2021). Yilmaz, C., Rezaei, M. & Sarkhosh, A. Environmental requirements and site selection. CABI , 225–246. (2021). https://doi.org/10.1079/9781789240764.0225 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 24 Apr, 2026 Reviews received at journal 15 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 06 Apr, 2026 Editor invited by journal 02 Apr, 2026 Submission checks completed at journal 30 Mar, 2026 First submitted to journal 27 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9112637","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":620758631,"identity":"78e6c769-620a-4126-87eb-276cc4b7553a","order_by":0,"name":"Mehdi Rezaei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACCQjFw8/Aw8AMZh6AihDUItkA18JMnBYGgwNoWnAC3dm9Dx/+qLGRMT5/9uDnwj02cnw38g8w/KhhkDFvwK7F7M5xYwOJY2k8ZjfykqVnPEszlryRzMDYc4yBR+YADi030tgkDNgOA7XwGEjzHDicuAGohYG3gYFHArsOkBb2Hwn//vMY958x/g3TwvgXvxY2hoNtB3gMGHLM4LYwE7CFWbKxL5lH4kaOmTXPAaBfzjw2OCxzTAKfFsaPP77Z2fMDHXab5wAwxI4nPnz4psbGHpcW7OAAIr5GwSgYBaNgFJADAFhQVPIovT7kAAAAAElFTkSuQmCC","orcid":"","institution":"Shahrood University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Rezaei","suffix":""},{"id":620758632,"identity":"46953f56-cf12-4fb2-870b-ad627f8a1f93","order_by":1,"name":"Parviz Heidari","email":"","orcid":"","institution":"Shahrood University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Parviz","middleName":"","lastName":"Heidari","suffix":""},{"id":620758633,"identity":"32c49497-4a4b-412f-a01c-b3d0b3578f0d","order_by":2,"name":"Stefanie Reim","email":"","orcid":"","institution":"Julius Kühn-Institut (JKI)","correspondingAuthor":false,"prefix":"","firstName":"Stefanie","middleName":"","lastName":"Reim","suffix":""}],"badges":[],"createdAt":"2026-03-13 09:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9112637/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9112637/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106784623,"identity":"6e45e7c4-54a6-43b6-97e8-53e69397d81d","added_by":"auto","created_at":"2026-04-13 12:20:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":450416,"visible":true,"origin":"","legend":"\u003cp\u003eDaily temperature patterns (minimum and maximum) during the 2023 growing season in two pomegranate cultivation regions of Shahrood, Iran: Toroud (warmer climate) on the left picture vs. Mayami (milder climate) on the right picture. MSN Weather data [2023]\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/76b83ebf57c056f876c30254.png"},{"id":106784628,"identity":"51af8524-d248-4772-a253-00d12e4a53b4","added_by":"auto","created_at":"2026-04-13 12:20:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1214376,"visible":true,"origin":"","legend":"\u003cp\u003eVisual comparison of aril quality in four pomegranate cultivars showing different levels of aril paleness (AP) disorder: Lower right picture: Externally healthy fruit with internally non-edible arils \u003cem\u003e‘DN’\u003c/em\u003e: ‘\u003cem\u003eDamavand’\u003c/em\u003e cultivar without paleness; \u003cem\u003e‘KN’\u003c/em\u003e: ‘\u003cem\u003eKashmar’\u003c/em\u003e cultivar with slight paleness; ‘\u003cem\u003eKW’\u003c/em\u003e: ‘\u003cem\u003eKashmar’\u003c/em\u003e cultivar with pronounced paleness; ‘\u003cem\u003eTW’\u003c/em\u003e: ‘\u003cem\u003eTorud’\u003c/em\u003e cultivar with severe aril paleness disorder.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/e729fbfd4ff4f13c059baecc.png"},{"id":106784624,"identity":"05fd7ac2-5ff0-446e-b3ed-9c070b1ecc8c","added_by":"auto","created_at":"2026-04-13 12:20:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74309,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of germination parameters and embryo viability in pomegranate cultivars with varying Aril Paleness (AP) susceptibility under control and GA3-treated conditions. Error bars represent standard deviation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/a0bf53ec4b4f3daddd1b042a.png"},{"id":106959801,"identity":"f33e81b7-8d6f-459a-aebc-07f789f52c2a","added_by":"auto","created_at":"2026-04-15 09:15:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3329241,"visible":true,"origin":"","legend":"\u003cp\u003eComparative seed anatomy and viability in pomegranate cultivars with (‘\u003cem\u003eTW’\u003c/em\u003e) and without (\u003cem\u003e‘DN’\u003c/em\u003e) aril paleness disorder. (A) Tetrazolium chloride (TTC)-stained embryos from \u003cem\u003e'TW'\u003c/em\u003e cultivar showing viable (red) versus non-viable (white) tissue (0.1% TTC, 12h incubation). (B) Longitudinal seed sections showing normal aril structure in \u003cem\u003e‘DN’\u003c/em\u003e and abnormal aril in ‘\u003cem\u003eTW’\u003c/em\u003e. (C) Histological tegmen abnormalities and progressive blackening of tegmen layers (arrows) in ‘\u003cem\u003eTW’\u003c/em\u003e seeds vs normal tissue in\u003cem\u003e‘DN’\u003c/em\u003e (Ci), (D) Distal tegmen blacking in ‘\u003cem\u003eTW’ \u003c/em\u003evs. normal tegmen color in ‘\u003cem\u003eDN’\u003c/em\u003e(Di). All images acquired using Olympus SZX10 stereo microscope.\u003cem\u003e ‘DN’\u003c/em\u003e: ‘\u003cem\u003eDamavand’\u003c/em\u003e cultivar without aril paleness; ‘\u003cem\u003eTW’\u003c/em\u003e: ‘\u003cem\u003eTorud’\u003c/em\u003e cultivar with severe aril paleness disorder.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/2a272d781e5bcb486e6f9b32.png"},{"id":106784627,"identity":"0ee75396-873f-4ac9-99b0-ebb5b36bbe45","added_by":"auto","created_at":"2026-04-13 12:20:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3193372,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal sections of arils from 'Torud' (\u003cem\u003eTW\u003c/em\u003e) cultivar showing embryonic developmental abnormalities in aril paleness disorder. A: Failed embryonic development: Complete absence of developed embryo (empty seed cavity). B: Embryo viability variations: Healthy, fully-formed embryo with showing cellular breakdown in tegmen coat (arrows indicate necrotic regions). C: Structural anomalies: Disorganized embryo positioning within seed cavity.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/51796ca0acc6c0492ffc8fa0.png"},{"id":106960491,"identity":"6cdc66bb-97da-4936-979f-1094f605d8b3","added_by":"auto","created_at":"2026-04-15 09:21:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6214516,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope (SEM) image of the discolored area of the seed coat (tegmen and mesotesta). Inset: Cellular degradation in the connection area of the sclereid cells of the tegmen and the mesotesta, and separation of the inner integument from the sclereid cells. The middle image and highlighted area show the selected region of the seed coat used for the SEM image, \u003cem\u003e‘DN’\u003c/em\u003e: \u003cem\u003e'Damavand'\u003c/em\u003ecultivar without aril paleness, ‘\u003cem\u003eKN\u003c/em\u003e: \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar without aril paleness, ‘\u003cem\u003eKW’\u003c/em\u003e: \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar with aril paleness, ‘\u003cem\u003eTW’\u003c/em\u003e: \u003cem\u003e'Torud'\u003c/em\u003e cultivar with aril paleness.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/1b07593e1ca80b098ec821b0.png"},{"id":106960538,"identity":"efd10d8b-684e-4a28-86f1-e8790458c635","added_by":"auto","created_at":"2026-04-15 09:21:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1468361,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope (SEM) images of inner sclereid cells of mesotesta in the seed coat of pomegranate cultivars with and without AP symptoms. The affected samples show thinner cell walls and smaller cell size, indicating structural degradation associated with aril paleness. The middle image and highlighted area show the selected region of the seed coat used for the SEM image. \u003cem\u003e‘DN’\u003c/em\u003e: \u003cem\u003e'Damavand'\u003c/em\u003ecultivar without aril paleness, ‘\u003cem\u003eKN’\u003c/em\u003e: \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar without aril paleness, ‘\u003cem\u003eKW’\u003c/em\u003e: \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar with aril paleness, ‘\u003cem\u003eTW’\u003c/em\u003e: \u003cem\u003e'Torud'\u003c/em\u003e cultivar with aril paleness.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/a94f7f460808d03a1a9c2c8e.png"},{"id":106963082,"identity":"20392440-6766-47e3-a4d8-bcdb082f8485","added_by":"auto","created_at":"2026-04-15 09:41:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19774307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9112637/v1/4f24b34a-a4ea-4a34-b3ae-27019b356ba4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seed Coat Degradation and Viability Loss in Pomegranate: The Hidden Cause of Aril Paleness Disorder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e) is an economically important fruit cultivated in subtropical and temperate regions with mild winters (Yilmaz et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Its high antioxidant content has generated substantial global interest, contributing to its growing market demand (Sarkhosh et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Pomegranate shrubs exhibit notable tolerance to drought and nutrient-poor soils, making them well-suited to arid and semi-arid environments. Major pomegranate-producing countries in the world include Iran, Turkey, India, Spain, and the United States (Yilmaz et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In Iran, most pomegranate orchards are located on the edges of desert regions characterized by mild winters (Ebrahimi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, in recent decades, coinciding with climate change, the incidence of physiological disorders such as fruit cracking and sunburn has increased significantly. Among these, a newly recognized physiological disorder referred to as \"Aril Paleness\" (AP) or \"Aril Browning\" has emerged as a major concern in many pomegranate-growing regions of Iran (Tabar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). AP-affected pomegranate fruits exhibit pale or browned arils with a dry, compromised internal texture, despite an outwardly healthy appearance. This not only reduces their appeal for fresh consumption but also makes them unsuitable for industrial processing, thereby impacting commercial value. Consumers often purchase pomegranates that appear healthy from the outsiden, only to find a low-quality product upon consumption. This has led to a decline in consumer trust and demand, creating significant challenges for quality control, particularly in export markets (Tabar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although several studies have proposed practical approaches to mitigate this physiological (Kavand et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Moradi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tadayon, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), none have proven consistently effective in reducing this disorder. One consistent finding across these studies is the existence of cultivar-specific susceptibility, with some genotypes showing higher resistance to the disorder than others (Kavand et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shivashankar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In affected fruits, arils fail to accumulate anthocyanin pigments, resulting in pale coloration. Additionally, the aril texture becomes sugary, slightly dehydrated, and develops an undesirable taste. When more than 50% of the arils are affected, the fruit is considered unfit for consumption. Numerous researchers have cited elevated temperatures during fruit development, linked to climate change, as key factor for the lack of pigment deficiency in pomegranate arils (Kavand et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Melgarejo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tabar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tadayon, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the precise physiological and anatomical mechanism underlying this disorder remain poorly understood.\u003c/p\u003e \u003cp\u003eTraditionally, the edible portion of the pomegranate fruit, the aril, has been described as a highly enlarged single cell. However, more recent anatomical studies by Melgarejo et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); Pujari and Rane (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), suggest that the aril comprises the entire seed structure, including the testa (outer integument), tegmen (inner integument), and the embryo with its cotyledons. The testa is notably thick, consisting of 5 to 18 layer of cells, while the tegmen is narrower and typically composed of 4 to 6 cell layers (Pujari \u0026amp; Rane, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Within the testa, two main components are distinguished: the fleshy, edible sarcotesta, and the thick-walled, hard, sclerotic mesotesta. During fruit ripening, cells in the testa undergo a considerable expansion, contributing to the development of the pulpy sarcotesta (Melgarejo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This tissue acts as a reservoir for water and bioactive compounds, contributing to the fruit\u0026rsquo;s sensory and nutritional qualities (Pujari \u0026amp; Rane, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The tegmen is also a multilayered structure, generally composed of an outer layer (4 to 5 cells thick) and an inner single-celled epidermis (Pujari \u0026amp; Rane, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Melgarejo-S\u0026aacute;nchez et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) demonstrated that changes in aril pigmentation correlates closely with fruit ripening stages, highlighting the importance of integument development in fruit quality. Based on these insights, our study investigates the anatomical abnormalities and viability of arils in pomegranate cultivars exhibiting varying degrees of aril paleness disorder (AP) different levels of disorder. We aimed to elucidate the underlying mechanisms of AP by assessing seed vigor, embryo viability, and the structure integrity of the surrounding integument tissues.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials\u003c/h2\u003e \u003cp\u003ePomegranate fruit samples were collected at commercial harvesting maturity from orchards in two distinct regions of Shahroud County, Iran: Mayami (36.2433\u0026deg; N, 55.3910\u0026deg; E) and Torud (36.4259\u0026deg; N, 55.0139\u0026deg; E). These sites were selected to represent two climatically different regions in Iran (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The samples were categorized according to the presence and severity of AP: \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e (\u003cem\u003e'Damavand'\u003c/em\u003e cultivar with normal arils, exhibiting no paleness), \u003cem\u003e\u0026lsquo;KN\u0026rsquo;\u003c/em\u003e (\u003cem\u003e'Kashmar'\u003c/em\u003e cultivar with normal arils, exhibiting slight paleness), \u0026lsquo;\u003cem\u003eKW\u0026rsquo;\u003c/em\u003e (\u003cem\u003e'Kashmar'\u003c/em\u003e cultivar exhibiting prominent AP), and \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e (\u003cem\u003e'Torud'\u003c/em\u003e cultivar exhibiting severe AP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e, \u0026lsquo;\u003cem\u003eKW\u0026rsquo;\u003c/em\u003e and \u003cem\u003e'KN'\u003c/em\u003e samples were sourced from the Mayami region of Shahroud County in Iran, an area with a relatively mild climate. The \u003cem\u003e'TW'\u003c/em\u003e samples originated from the Torud region in Iran known for its warmer climate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fruits were harvested at full maturity, based on visual assessment of skin color and fruit size. From each cultivar, 12\u0026ndash;25 fruits were randomly collected from three trees within commercial orchard.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeed Viablity Tests\u003c/p\u003e \u003cp\u003ePomegranate seeds were extracted from three randomly selected fruits per cultivar. Following extraction, the seeds were cleaned with water to remove any remaining pulp, after which they were air-dried at an ambient temperature of approximately 25\u0026deg;C for seven days. The seeds were surface-sterilized by immersing them in a 1% sodium hypochlorite (NaOCl) solution for 10 minutes, followed by three rinses with sterile distilled water. A gibberellic acid (GA\u003csub\u003e3\u003c/sub\u003e) treatment was also employed to promote germination. The seeds were imbibed by being soaked in either a 500 mg/L GA\u003csub\u003e3\u003c/sub\u003e solution or sterile distilled water (control) for 12 hours under dark conditions. Following imbibition, groups of 25 seeds were transferred to sterile Petri dishes (90 mm diameter) containing two layers of Whatman\u0026reg; No. 1 filter paper that had been moistened with sterile distilled water. The experimental design was completely randomized, with four replicates per treatment. Petri dishes were sealed with Parafilm to minimize moisture loss and incubated in a controlled-environment germination chamber maintained at a constant temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a photoperiod of 16 hours. Germination was monitored daily for a duration of 50 days, starting from the third week after sowing. A seed was considered germinated when the radicle emerged by at least 2 mm. Germination parameters were calculated as follows:\u003c/p\u003e \u003cp\u003eGermination percentage (GP) = (Ng / Nt) \u0026times; 100, where Ng is the number of germinated seeds and Nt is the total number of seeds tested.\u003c/p\u003e \u003cp\u003eGermination vigor (GV) = Σ (germinated seeds on day n \u0026times; n) / total seeds. (higher values indicate greater vigor).\u003c/p\u003e \u003cp\u003eA tetrazolium test was also conducted to assess the viability of embryo and cotyledon tissues. The seeds were initially pre-conditioned by soaking them in sterile distilled water for 24 hours to soften the tissue. Following imbibition, the pomegranate seeds were carefully dissected using a sterile scalpel under aseptic conditions. The embryos and cotyledons were then excised from the seeds in an aqueous environment using a fine needle to minimize mechanical damage. Excised tissues were then immersed in a 0.1% (w/v) tetrazolium chloride (2,3,5-triphenyltetrazolium chloride; TTC) solution prepared in phosphate buffer (50 mM, pH 7.0) and incubated in the dark at 25\u0026deg;C for 12 hours. During incubation, viable tissues reduce the colorless TTC to red-colored formazan. Following incubation, tissues were rinsed three times with distilled water to remove excess TTC. The extent and intensity of formazan staining in the embryo and cotyledon tissues were visually assessed and scored under a stereomicroscope (Olympus SZX10). Thirty seeds from three separate fruits were evaluated per treatment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological analysis\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e-Seed Coat Abnormality\u003c/h3\u003e\n\u003cp\u003eTo examine the internal structure of the seed coat, fresh pomegranate seeds were extracted in an aqueous solution to prevent desiccation. The seeds were bisected with a sterile scalpel and the embryo and cotyledons were carefully removed. The internal surface of the seed coat was then examined and photographed under a stereomicroscope (Olympus SZX10) to assess its cellular structure and identify any abnormalities. A total of thirty seeds from three separate fruits were evaluated, with three fruits taken from each pomegranate cultivar.\u003c/p\u003e\n\u003ch3\u003e- Scanning Electron Microscopy and Energy-Dispersive Spectra\u003c/h3\u003e\n\u003cp\u003eBased on stereomicroscope observations of affected tissues within the hard seed coat, the tegmen and the mesotesta, samples of the hard seed coat were prepared for scanning electron microscopy (SEM). The samples were initially dried in a desiccator containing silica gel until they reached a constant weight to ensure complete dehydration. The dried samples were then mounted on aluminium stubs using carbon tape and sputter-coated with a thin layer of gold (approximately 20 nm) in a vacuum using a tabletop sputter coater (Cressington 108auto) to enhance conductivity and prevent charging during imaging. Images were acquired at various magnifications (ranging from 500x to 2000x) using a field emission scanning electron microscope (FESEM, Zeiss Sigma 300-HV, Germany) operated at an accelerating voltage of 5 kV. In addition, elemental analysis was performed on the surface of each sample using energy-dispersive X-ray spectroscopy (EDS, Oxford Instruments) which was integrated with the FESEM. Three samples per cultivar were analyzed, and EDS spectra were collected from multiple points on each sample to ensure representative elemental composition data.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e-Seed Viability Tests\u003c/h2\u003e \u003cp\u003eThe germination study revealed clear disparities in germination potential among the pomegranate cultivars. A striking correlation was observed between AP and reduced germination percentage and rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The \u003cem\u003e'DN'\u003c/em\u003e cultivar, which exhibited no AP, demonstrated the highest germination percentage, highlighting its robust seed viability. Conversely, the \u003cem\u003e'TW'\u003c/em\u003e and \u003cem\u003e'KW'\u003c/em\u003e cultivars with pronounced AP, showed markedly reduced germination, indicating a detrimental effect of the paleness on seed quality. While the \u003cem\u003e'KN'\u003c/em\u003e cultivar with slight paleness displayed intermediate germination, it was still significantly lower than that of the \u003cem\u003e'DN'\u003c/em\u003e cultivar (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interestingly, the application of gibberellic acid (GA\u003csub\u003e3\u003c/sub\u003e) did not consistently improve germination rates across all cultivars. In fact, a slight reduction in germination rates was observed in the \u003cem\u003e'DN'\u003c/em\u003e cultivar under GA\u003csub\u003e3\u003c/sub\u003e treatment, though the high variability of the results makes this inconclusive. As can been seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the \u003cem\u003e'DN'\u003c/em\u003e cultivar exhibits the highest germination rate under both control and GA3-treated conditions. The control group of \u003cem\u003e'DN'\u003c/em\u003e has a germination rate of approximately 17 seeds per week, while the GA3-treated group shows a rate of around 13 seeds per week. The \u003cem\u003e\u0026lsquo;KN\u0026rsquo; cultivar\u003c/em\u003e, exhibits a lower germination rate compared to the \u003cem\u003e'DN'\u003c/em\u003e cultivar. The \u003cem\u003e\u0026lsquo;KW\u0026rsquo; and 'TW'\u003c/em\u003e cultivars exhibit even lower germination rates than \u0026lsquo;\u003cem\u003eKN\u0026rsquo;\u003c/em\u003e cultivar. Despite the significant differences observed in germination tests among cultivars with varying degrees of AP disorder, the tetrazolium viability tests of embryo revealed that the examined pomegranate cultivar with extreme AP, \u003cem\u003e'TW'\u003c/em\u003e, still maintained over 67% healthy and viable embryonic tissue (Fg. 3, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, a statistically significant difference was found between cultivars exhibiting AP and those with normal arils; the \u003cem\u003e'KN'\u003c/em\u003e and \u003cem\u003e'DN'\u003c/em\u003e cultivars showed over 98% viable embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e-Histological analysis\u003c/h2\u003e \u003cp\u003eA histological examination was conducted on seeds from each cultivar using longitudinal sections to observe the internal structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e cultivar, over 90% of the seeds exhibited a distinct blacking region at the distal end of the inner integument (tegmen) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, D), whereas in \u0026lsquo;\u003cem\u003eDN\u0026rsquo;\u003c/em\u003e no blacking of the tegmen was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Ci and Di). Around 20% of these seeds showed tissue degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C), and in a few arils, the embryo was not formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This blackening in tegmen was also visible in 58% of the seeds from the \u0026lsquo;\u003cem\u003eKW\u0026rsquo;\u003c/em\u003e cultivar, whereas only 10% of \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e and \u0026lsquo;\u003cem\u003eKN\u0026rsquo;\u003c/em\u003e seeds showed this discoloration in the inner tegmen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of SEM images of seed coats from different pomegranate cultivars revealed a clear relationship between AP and the structural integrity of the seed coat (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The \u003cem\u003e'DN'\u003c/em\u003e cultivar, which exhibited no AP, showed a healthy and intact seed coat structure, characterized by well-organized sclereid cells and a distinct interface with the inner integument (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, picture \u003cem\u003eDN\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, cultivars with AP (\u0026lsquo;\u003cem\u003eKW\u0026rsquo;\u003c/em\u003e and \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e) displayed marked degradation and disruption of the seed coat, particularly in the sclereid cell layer and at the interface with the inner integument. These changes were evident as collapsed cells and structural disorganization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, pictures \u003cem\u003eKW\u003c/em\u003e and \u003cem\u003eTW\u003c/em\u003e). The \u003cem\u003e'KN'\u003c/em\u003e cultivar, showing mild AP symptoms, exhibited an intermediate level of degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, picture \u003cem\u003eKN\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThese results suggest that AP may be associated with processes that compromise the development or maintenance of seed coat structure, potentially reducing seed viability. Supporting this, Fig.\u0026nbsp;7 shows that seed coat cells in healthy cultivars had thicker, more intact cell walls compared to those affected by AP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDN\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure 7. Scanning electron microscope (SEM) images of inner sclereid cells of mesotesta in the seed coat of pomegranate cultivars with and without AP symptoms. The affected samples show thinner cell walls and smaller cell size, indicating structural degradation associated with aril paleness. The middle image and highlighted area show the selected region of the seed coat used for the SEM image. \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e: \u003cem\u003e'Damavand'\u003c/em\u003e cultivar without aril paleness, \u0026lsquo;\u003cem\u003eKN\u0026rsquo;\u003c/em\u003e: \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar without aril paleness, \u0026lsquo;\u003cem\u003eKW\u0026rsquo;\u003c/em\u003e: \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar with aril paleness, \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e: \u003cem\u003e'Torud'\u003c/em\u003e cultivar with aril paleness.\u003c/p\u003e \u003cp\u003e-EDS analysis\u003c/p\u003e \u003cp\u003eComparative EDS (Energy Dispersive X-ray Spectroscopy) analysis of seed coats from different pomegranate cultivars revealed distinct elemental differences associated with varying levels of AP susceptibility (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this study, elemental variations observed between cultivars provide further evidence of physiological differences related to seed coat integrity and the occurrence of AP. The highly susceptible cultivar \u003cem\u003e'TW'\u003c/em\u003e showed a 5.6% reduction in oxygen concentration (41.72 wt%) compared to the resistant cultivar \u003cem\u003e'DN'\u003c/em\u003e (44.05 wt%), along with a corresponding increase in carbon levels (\u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e: 57.99 wt% vs. \u0026lsquo;\u003cem\u003eDN\u0026rsquo;\u003c/em\u003e: 55.23 wt%). Calcium levels were remarkably lower in \u0026lsquo;TW\u0026rsquo; (0.05 wt%) than in \u0026lsquo;DN\u0026rsquo; (0.16 wt%), reflecting a 68.7% decrease. Potassium content was also reduces in \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e (0.17 wt%) compared to \u0026lsquo;\u003cem\u003eDN\u0026rsquo;\u003c/em\u003e (0.32 wt%), representing a 47% decline. Interestingly, sulfur was uniquely detected in the moderately affected \u003cem\u003e'KW'\u003c/em\u003e cultivar (0.10 wt%), possibly indicating stress-induced accumulation of sulfur-containing metabolites. Magnesium was exclusively present in \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e (0.06 wt%), suggesting atypical cationic substitution within lignified strata. Together, these elemental deviations point to cultivar-specific differences in seed coat composition and integrity, elucidating the physiological mechanisms of impaired nutrient transport and weakened cell adhesion in AP-susceptible tissues.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative elemental composition of seed coats across pomegranate cultivars with varying AP susceptibility\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e )wt%(\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lsquo;\u003cem\u003eKN\u0026rsquo;\u003c/em\u003e (wt%(\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lsquo;\u003cem\u003eKW\u0026rsquo;\u003c/em\u003e (wt%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e (wt%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eND\u0026thinsp;=\u0026thinsp;Not Detected (\u0026plusmn;\u0026thinsp;0.01 wt% detection limit). \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e: 'Damavand' cultivar without paleness, KN: 'Kashmar' cultivar without paleness, KW: 'Kashmar' cultivar with paleness, TW: 'Torud' cultivar with paleness\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated anatomical and physiological traits linked to aril paleness disorder (AP) in four pomegranate cultivars with varying susceptibility. Seed viability and seed coat structure were analyzed to identify factors that may explain differences in AP resistance. Our findings provide robust evidence that AP disorder in pomegranates is primarily driven by critical impairments in embryo development, compounded by structural deficiencies in the seed coat.\u003c/p\u003e\n\u003ch3\u003eEmbryo Viability as the Primary Driver of Aril Paleness Disorder (AP)\u003c/h3\u003e\n\u003cp\u003eThe viability tests revealed that aril paleness disorder (AP) in pomegranates is strongly associated with a drastic reduction in seed germination capacity and a high incidence of non-viable embryos in affected fruits. In particular, the highly susceptible cultivar \u0026lsquo;Torud\u0026rsquo; (TW) exhibited a germination rate of only 4%, compared to 45% in the resistant cultivar \u0026lsquo;Damavand\u0026rsquo; (DN), despite both being cultivated under identical management practices. These findings are consistent with previous research by Shivashankar et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), who also identified the seed as the primary origin of aril paleness disorder.\u003c/p\u003e \u003cp\u003eSeveral factors may contribute to impaired embryo development and reduced seed viability, including cultivar specific susceptibility or environmental stresses such as elevated temperatures and drought (Faraji and Karami, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is supported by the highly susceptible cultivar \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e with most pronounced seed coat abnormalities, which is growing in the warmer regions like Toroud. In addition, nutrient imbalances and disruptions in phytohormone signaling, which is normally mediated by the embryo to regulate fruit development, may also play a role. Genetic predisposition appears to be a key factor as well, with resistant cultivars like \u0026lsquo;\u003cem\u003eDN\u0026rsquo;\u003c/em\u003e likely possessing inherent mechanisms that support normal embryo development even under stress(Mohammad Kavand et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe connection between impaired embryo development and aril degradation is multifaceted. A non-functional embryo loses its role as a metabolic sink, leading to the accumulation of sugars and other metabolites in the aril tissue. This metabolic imbalance disrupts anthocyanin biosynthesis, which is resulting in pale coloration, and increases oxidative stress, which in turn activates browning-related enzymes. Supporting this, Meena et al. (2021) reported that necrosis in the innermost mesocarp tissues during endocarp hardening coincides with the onset of aril browning. Altogether, the physiological collapse of the embryo and surrounding tissues initiates a cascade of metabolic and structural deterioration that culminates in the visible symptoms of AP(Shivashankar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSeed Coat Structure and Elemental Integrity as Key Modulators of AP Expression\u003c/h2\u003e \u003cp\u003eFurthermore, our study highlights severe degradation of sclereid cells and inner integument layers in affected AP pomegranates, evidenced by SEM images showing cell separation. This structural damage directly impedes solute transfer to developing arils and compromises the seed coat's protective function, making it vulnerable to stress. The structural integrity of the seed coat, particularly the sclerotic mesotesta, is vital for nutrient transport. Our EDS findings of calcium depletion in AP-susceptible cultivars (\u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e: 0.05 wt% vs. \u0026lsquo;\u003cem\u003eDN\u0026rsquo;\u003c/em\u003e: 0.16 wt%) further align with Pujari \u0026amp; Rane's (2015) work, which established calcium's role in the lignification and rigidity of mesotesta cells. This suggests that AP may involve climate-disrupted calcification of these sclerotic layers, impairing vascular transport to the arils. The coincidence of the 45-day post-anthesis sclerification window (Pujari \u0026amp; Rane, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) with our observed temperature sensitivity period is critical, strongly suggesting that AP mitigation strategies should target early fruit development through timely calcium-boron supplementation to ensure proper sclerification and overall seed coat health, thereby protecting embryo development and maintaining fruit quality.\u003c/p\u003e \u003cp\u003eIn addition, EDS (Energy Dispersive X-ray Spectroscopy) revealed an altered C:O ratio in \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e (1.85) compared to \u0026lsquo;\u003cem\u003eDN\u0026rsquo;\u003c/em\u003e (1.67), suggesting degradation of oxygen-abundant pectic polysaccharides in the cell wall matrix (Anderson, 2019), consistent with middle lamella disintegration observed in SEM imagery. This likely disrupts pectate cross-linking essential for cell wall cohesion (Wdowiak et al., 2024). As EDS is a well-established method for detecting nutritional and elemental changes in plant tissues (Kopittke et al., 2020; van Der Ent et al., 2018; Wyroba et al., 2015), these findings provide further evidence that cell wall integrity in AP-affected seed coats is severely compromised. Ultimately, both embryo viability and seed coat structure must be considered in a comprehensive understanding of AP, as both elements appear to interact and amplify the disorder under unfavorable environmental conditions.\u003c/p\u003e \u003cp\u003eThese findings suggest that AP mitigation should focus on early fruit development, particularly during the sclerification window. Targeted nutrient management, including calcium and boron supplementation, may support proper seed coat formation and protect embryo development, thereby improving overall fruit quality and resistance to AP. Cultivar selection also plays a crucial role, and breeding programs may benefit from prioritizing genetic lines with inherent embryo resilience and seed coat integrity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study demonstrates that aril paleness disorder in pomegranates is primarily associated with impaired embryo development and compromised seed coat integrity. Both factors interact synergistically, especially under environmental stress, to promote AP expression. However, the limited number of cultivars examined and the lack of multi-site environmental replication suggest caution in generalizing these findings. Future research should focus on controlled environmental studies and molecular investigations of embryo development to confirm and expand upon these insights. Early-stage nutrient interventions and cultivar selection nevertheless emerge as promising strategies to mitigate this economically significant disorder and improve overall fruit quality.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAril paleness\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003e'Damavand'\u003c/em\u003e pomegranate cultivar with normal arils\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar with normal arils\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eexhibiting slight paleness\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003e'Kashmar'\u003c/em\u003e cultivar exhibiting prominent AP\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003e'Torud'\u003c/em\u003e cultivar exhibiting severe AP\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScanning electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGA3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGibberellic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGermination percentage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGermination vigor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e5-triphenyltetrazolium chloride\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnergy-dispersive X-ray spectroscopy.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/h2\u003e\n\u003cp\u003eAll plant materials used in this study were obtained with proper authorization. Pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) samples were collected from three privately-owned commercial orchards in Mayami (36.24\u0026deg;N, 55.39\u0026deg;E) and Torud (36.62\u0026deg;N, 55.01\u0026deg;E), Shahroud, Iran, following agreements with the owners (Mr. Abedian and Mr. Mirii). Voucher specimens (POM-2025-001 to POM-2025-004) have been deposited in the Herbarium Lab., Agriculture Faculty, Sharood University of Technology, Shahrood, Iran.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eClinical trial number\u003c/h2\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;Declaration:\u003c/strong\u003e This work was supported by the Center for International Scientific Studies \u0026amp; Collaboration (CISSC), Ministry of Science, Research and Technology of Iran.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAuthors' contributions: MR and PH conducted data collection and experiments. MR supervised the project and wrote the manuscript. MR, PH, and ER contributed to conceptualization and data interpretation. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll study data are included in the manuscript. Additional datasets are available from the corresponding author upon reques\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFaraji, S. \u0026amp; Karami, S. Spatial distribution of pomegranate aril paleness and its relationship with some environmental and non-environmental factors using geographic information system (GIS). \u003cem\u003eIran. J. Hortic. Sci.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e (3), 495\u0026ndash;513. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22059/ijhs.2024.372626.2156\u003c/span\u003e\u003cspan address=\"10.22059/ijhs.2024.372626.2156\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahimi, M. Production and supply of pomegranate in Iran. \u003cem\u003eЕкономіка АПК\u003c/em\u003e (7), 121\u0026ndash;125. (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKavand, M., Arzani, K., Barzegar, M. \u0026amp; Mirlatifi, M. Effects of sunscreen, kaolin application, fruit thinning and supplementary irrigation on the aril browning disorder of Pomegranate cv.Malase Torshe Saveh. \u003cem\u003eSeed Plant. Prod. J.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e (1), 85\u0026ndash;112 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKavand, M., Arzani, K., Barzegar, M. \u0026amp; Mirlatifi, M. Identification of the tolerant pomegranate genotypes for the aril browning or aril paleness disorder. I International Conference and X National Horticultural Science Congress of Iran (IrHC2017) 1315, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelgarejo-S\u0026aacute;nchez, P. et al. Pomegranate variety and pomegranate plant part, relevance from bioactive point of view: A review. \u003cem\u003eBioresources Bioprocess.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 1\u0026ndash;29 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelgarejo, P., Mart\u0026iacute;nez-Nicol\u0026aacute;s, J. J., N\u0026uacute;\u0026ntilde;ez-G\u0026oacute;mez, D., Almansa, M. S. \u0026amp; Legua, P. Accurate Botanical Nomenclature: Pomegranate and the \u0026lsquo;Aril\u0026rsquo;Misconception. \u003cem\u003eFoods\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (2), 201 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradi, S. et al. Fruit quality, antioxidant, and mineral attributes of pomegranate cv. Ghojagh, influenced by shading and spray applications of potassium sulfate and sodium silicate. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 14831 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePujari, K. \u0026amp; Rane, D. A. Concept of seed hardness in pomegranate - i) anatomical studies in soft and hard seeds of 'muskat' pomegranate. \u003cem\u003eActa Hort.\u003c/em\u003e \u003cb\u003e1089\u003c/b\u003e, 97\u0026ndash;104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.17660/ActaHortic.2015.1089.11\u003c/span\u003e\u003cspan address=\"10.17660/ActaHortic.2015.1089.11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkhosh, A., Yavari, A. M. \u0026amp; Zamani, Z. \u003cem\u003eThe pomegranate: botany, production and uses\u003c/em\u003e (CAB International, 2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cabidigitallibrary.org/doi/abs/\u003c/span\u003e\u003cspan address=\"https://www.cabidigitallibrary.org/doi/abs/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1079/9781789240764.0000\u003c/span\u003e\u003cspan address=\"10.1079/9781789240764.0000\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShivashankar, S., Singh, H. \u0026amp; Sumathi, M. Aril browning in pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) is caused by the seed. \u003cem\u003eCurrent Sci. (00113891)\u003c/em\u003e, \u003cb\u003e103\u003c/b\u003e(1). (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabar, S. M., Tehranifar, A., Davarynejad, G. H., Nemati, S. H. \u0026amp; Zabihi, H. R. Aril Paleness, New Physiological Disorder in Pomegranate Fruit (Punica granatum): Physical and Chemical Changes during Exposure of Fruit Disorder: Physical and Chemical Changes during Exposure of Fruit Disorder. \u003cem\u003eHortic. Environ. Biotechnol.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e (4), 300\u0026ndash;307 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadayon, M. S. Effect of foliar nutrition with calcium, boron, and potassium on amelioration of aril browning in pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e cv.\u0026lsquo;Rabab\u0026rsquo;). \u003cem\u003eJ. Hortic. Sci. Biotechnol.\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e (3), 372\u0026ndash;382 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYilmaz, C., Rezaei, M. \u0026amp; Sarkhosh, A. Environmental requirements and site selection. \u003cem\u003eCABI\u003c/em\u003e, 225\u0026ndash;246. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/9781789240764.0225\u003c/span\u003e\u003cspan address=\"10.1079/9781789240764.0225\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Punica granatum, seed viability, embryo health, scanning electron microscopy (SEM), physiological disorder","lastPublishedDoi":"10.21203/rs.3.rs-9112637/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9112637/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePomegranate aril paleness (AP), a physiological disorder linked to climate change, causes pale, desiccated arils in otherwise healthy fruits, reducing marketability. To investigate the anatomical and physiological underpinnings of AP, this study examined four pomegranate cultivars exhibiting varying degrees of susceptibility: the resistant cultivar \u003cem\u003e'Damavand'\u003c/em\u003e (\u003cem\u003eDN\u003c/em\u003e), the moderately affected cultivars \u003cem\u003e'Kashmar'\u003c/em\u003e (\u003cem\u003eKN\u003c/em\u003e and \u003cem\u003eKW\u003c/em\u003e), and the severely affected cultivar \u003cem\u003e'Torud'\u003c/em\u003e (\u003cem\u003eTW\u003c/em\u003e). Seed viability was assessed using germination tests and tetrazolium staining, while structural abnormalities in the seed coat were assessed through histological analysis and scanning electron microscopy (SEM). The results revealed a strong association between AP severity and reduced germination rates, with germination rates ranging from 45% in the resistant cultivar \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e to just 4% in the severely affected cultivar \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e. Interestingly, tetrazolium tests indicated that a large proportion of embryos remained viable despite severe AP symptoms, ranging from 67% in \u0026lsquo;\u003cem\u003eTW\u0026rsquo;\u003c/em\u003e to 98% in \u003cem\u003e\u0026lsquo;DN\u0026rsquo;\u003c/em\u003e. Microscopic analyses further demonstrated substantial structural degradation in the seed coats of AP-affected cultivars, including blackened regions, tissue separation, and reduced cellular density in the inner seed coat layers. Taken together, the findings highlight that AP is associated with specific anatomical abnormalities in the seed coat and tegmen, which may underlie or exacerbate the physiological manifestations of the disorder.\u003c/p\u003e","manuscriptTitle":"Seed Coat Degradation and Viability Loss in Pomegranate: The Hidden Cause of Aril Paleness Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 12:20:16","doi":"10.21203/rs.3.rs-9112637/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T10:21:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T12:43:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T18:04:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71685441819378460548638755260581876917","date":"2026-04-07T15:53:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46991302032340682804053600069796885966","date":"2026-04-07T12:14:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T05:55:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T01:11:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-03T02:38:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-30T14:01:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-27T07:39:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"81224ba8-cc07-4621-b91f-31cf0305abe3","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T10:21:40+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66062181,"name":"Biological sciences/Physiology"},{"id":66062182,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-13T04:53:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 12:20:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9112637","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9112637","identity":"rs-9112637","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}