In this study, high-throughput sequencing technology was used to reveal the diversity of endophytic bacterial communities in the dimorphic seeds of S. glauca obtained from the same natural environment. Our findings demonstrated that seed dimorphism had little impact on the diversity and richness of endophytic bacterial communities in brown and black seeds, but significantly different relative abundances of the endophytic bacterial taxa were detected in brown and black seeds of S. glauca.
Many studies have shown that the seed dimorphism of Suaeda spp. usually associated with differences in seed shape, seed size, seed coat color [2, 6–8, 11–12, 14–15], seed coat structure , seed germinability [7, 11, 12, 15] and seed phytochemical properties [16–18]. In this study, we observed that seed coat structure and seed phytochemical properties of brown and black seeds of S. glauca significantly differed. Our results revealed that black seeds of S. glauca had two layers of seed coat, including a layer of hard, cuticle exotesta and a layer of soft, membranous endotesta compared to single-layer membranous testa in brown seeds. A similar result has also been reported in Borszczowia aralocaspica (S. aralocaspica) . Seed coat acts as a modulator between seed and environment and can regulate gaseous exchange and water imbibition [40, 41]. A previous study has indicated that black seeds of S. glauca had an intermediate physical dormancy and exhibited a low germination percentage, but it was water-permeable . Brits et al. [42, 43] demonstrated that the intact hard testa may partially reduce oxygen diffusion to the embryo, contribute to hypoxic constraints. Brits and Manning  found the seeds of Leucospermum cordifolium have also two layers of seed coat (exotesta and endotesta), and exhibit water-permeable and oxygen-impermeable, which named as “anoxia PY (physical dormancy)”. Besides, Wang et al.  reported that the seed coat of black seeds of S. salsa contains a high content of waxes compared to brown seeds. These results implied that the difference in both structure and chemical composition of seed coat leads to differences in oxygen exchange capacity between black and brown seeds of S. glauca. The black seeds may rather have limited capacity for gas exchange compared to brown seeds. Interestingly, Tegtmeier et al.  found that oxygen availability can influence colonization patterns of microbes in the gut microbiota.
Our results revealed that the content of soluble protein, soluble starch and soluble sugar was significantly higher in extracts obtained from brown seeds than those of black seeds; in contrast, the content of fat in brown seeds was lower than black seeds. The different abilities of nutrition accumulation in dimorphic seeds have also been reported in S. salsa [17, 36] and S. aralocaspica [16, 18]. For example, Song et al.  found that the content of soluble sugar, soluble protein, total nitrogen, total phosphorus and inorganic ions (K+, Na+, K+/Na+) in brown seeds were significantly higher than those of black seeds in S. aralocaspica. In addition, we also detected higher content of total phenols in brown seeds compared to that of black seeds. Similar result was also reported in S. salsa . Overall, these results suggested that there were significant differences in seed phytochemical properties between the dimorphic seeds of S. glauca. Interestingly, numerous studies have determined that the compositions of seed endophytic microbiota have been influenced the seed phytochemical traits [34, 35].
In the present study, alpha-diversity indices were used to evaluate the seed endophytic bacterial community richness and diversity. The results showed that no significant differences in alpha-diversity indices were found between brown seeds and black seeds. It was quite surprising that the significant differences in the seed coat structure and seed phytochemical characteristics between brown and black seeds had little impact on the diversity and richness of endophytic bacterial communities in the dimorphic seeds. Similar result has also reported by Zhang et al. , who found five rice genotypes have little impact on the diversity and richness of endophytic bacteria.
In the present study, 9 prokaryotic phyla were observed, of which Proteobacteria, Firmicutes and Actinobacteria were dominant. These above-mentioned phyla have also been reported as dominant endophytes of other plant seeds [46, 47]. Meanwhile, Kushneria, Halomonas, Bacillus, Marinilactibacillus, Rhodococcus, Ralstonia, Pelomonas and Bradyrhizobium were described as the high relative abundant genera, of them, Kushneria, Halomonas and Bacillus were the core endophytic bacterial community. Interestingly, Kushneria, Halomonas and Bacillus have also reported as dominant endophytes from roots of halophytes, such as Salicornia rubra, Sarcocornia utahensis and Allenrolfea occidentalis . Previous studies have revealed that Kushneria strains were isolated mostly from saline environments , endosphere of halophyte Arthrocnemum macrostachyum  and Avicennia germinans , phyllosphere of halophyte Avicennia germinans  and rhizosphere of halophyte Saccharum spontaneum . Some members of the genus Kushneria reported having plant growth-promoting activities, including siderophore production, indolacetic acid (IAA) production, nitrogen fixation and phosphate solubilization [50, 54]. Halomonas and Kushneria are closely related, and were grouped in the same genus in the past . Many Halomonas sp. exhibit salt tolerance and can improve plant growth under salt stress conditions [48, 55–57]. Bacillus is common genera among the endosphere niche of diverse plants, where they play an important role in plant protection and growth stimulation [58, 59]. The results suggested that these core taxa may play an important role on the seed endosphere of halophyte S. glauca, and these taxa can assist the plant to resistance stress environments. Besides, the Venn diagram revealed that greater taxa presented in brown seeds, and also had high heterogeneity within the bacterial communities compared to black seeds (Fig. 5). One possible explanation was that brown seed with single layer membraneous seed coat and abundant nutrients that could contribute to colonize microorganisms present in the carposphere of utricles, and easily susceptible to the carposphere environment. Recent studies have shown that seed bacterial endophytes may also originate from the phyllosphere, anthosphere and carposphere [26, 60].
Based on alpha diversity analysis, PCoA analysis, and hierarchical clustering tree results, seed dimorphism had no significant impact on diversity indices as a whole, it influenced significantly the relative abundance of endophytic bacterial taxa between brown and black seeds. Our comprehensive comparison revealed that the relative abundances of endophytic bacterial communities of dimorphic seeds were significantly different from each other at phylum, class, order, family and genus level. At the phylum level, we observed 9 identified phyla, one phyla of the three dominant phyla exhibited statistically different. Interestingly, the relative abundance of Actinobacteria was higher in brown seeds than in black seeds, which means Actinobacteria may be enriched in brown seeds. This might attribute to brown seeds with single layer membranous seed coat and fast germinability were easily susceptible to soil-borne pathogens compared to black seeds, while Actinobacteria may protect brown seeds against pathogens and promotes plant growth [61, 62]. Gripenberg et al.  found that there was a potential trade-off between seed chemical and mechanical defense, polyphenols as one of the most common seed defenses, which are most likely to be present in large seeds with short seed dormancy and low investment in mechanical seed defense. Compared to black seeds with high investment in mechanical seed defense (two layers seed coat, including hard exotesta and soft, membranous endotesta), brown seeds had a high level of phenolic content. Hence, we speculated that a high abundance of Actinobacteria, combined with high levels of total phenols, can protect brown seeds from pathogens in the soil seed bank.
At the genus level, 5 genera of the 8 dominant genera possessed statistically significant differences between brown seeds and black seeds. Rhodococcus, Ralstonia, Pelomonas, Bradyrhizobium and Marinilactibacillus exhibited significantly difference between the two groups. Notably, we found that Rhodococcus, Ralstonia, Pelomonas and Bradyrhizobium tend to be enriched in brown seeds, and present in high proportion compared to black seeds. Especially, our results revealed that Rhodococcus erythropolis, Ralstonia solanacearam, Pelomonas (species unclassified) and Bradyrhizobium elkanii, were the dominant species in brown seeds (Fig. S2). Rhodococcus have been found living in close association with various plant parts, such as rhizosphere , phyllosphere [65, 66] and endosphere [67–70]. R. erythropolis can colonize plant roots , and also prevent plant disease by degrading N-acyl-homoserine lactone signaling molecules . Moreover, several members of the genus Rhodococcus also show plant growth-promoting activities, including ACC deaminase, IAA production, siderophore production and phosphate solubilization [72–75]. Some strains of the genus Pelomonas detected in the endosphere of Typha angustifolia , and reported to fix nitrogen . Bradyrhizobium, a genus of Gram-positive that initially proposed as a group of slow-growing, alkaline-producing root nodule nitrogen-fixing bacteria . B. elkanii isolated from the root nodules of Acacia confusa, exhibit the nitrogen-fixing ability and can enhance the growth and root development of A. confuse . Numerous studies revealed that endophytic bacteria can improve plant fitness by enhancing nutrient mobilization, nitrogen fixation, phosphate solubilization and conferring resistant against pathogens [27, 80]. Thus, we speculated that brown seeds harbor a large number of microorganisms with plant growth-promoting (PGP) traits, which contribute to the establishment and development of seedling of the brown seeds, since brown seeds without dormancy behavior, were the main source of early spring seedling of S. glauca . In addition, we also detected strains of Ralstonia in brown seeds, such as R. solanacearam, which is an important soil-borne plant pathogen with . Taken together, it seemed that brown seeds served not only as hosts for diverse plant-probiotic bacterial strains but also for putative opportunistic pathogenic bacteria.
In our study, compared to the endophytic microbiota of brown seeds, we found that Marinilactibacillus tends to be enriched in black seeds, and had higher proportions. Remarkably, Marinilactibacillus has also firstly reported as one of the most abundant genera in the endosphere of halophyte Halimione portulacoides . Previous study revealed that Marinilacibacillus piezotolerans was a facultatively anaerobic lactobacillus [83, 84]. The results implied that Marinilactibacillus may adapt the inner hypoxia environment of black seeds, since two layers of the seed coat of black seeds prevent gas-exchange. Truyens et al.  found that selection of seed endophytes partly relies on the bacterial properties, and only bacteria with competitive and adaptive colonization characteristics can inhabit the seeds . The functional differentiation of the seed endophytic bacterial communities was manifested by the differential enrichments of pentose phosphate pathway, pentose phosphate pathway (non-oxidative branch) and gondoate biosynthesis (anaerobic) between brown and black seeds. We found that these metabolic pathways also tend to be enriched in black seeds and had higher proportions compared to brown seeds. A previous study has shown that the pentose phosphate pathway (PPP) can antagonize oxidative stress in hypoxia by producing NADPH and ribose-5P . Interestingly, Fidalgo et al.  found that Marinilactibacillus spp. isolates tested positive for cellulolytic, proteolytic and xylanolytic enzymatic activities. Strain B. gibsonii (Fig. S2) was also highly enriched species in black seeds, which was an efficient alkaline pectinase producer . Together, the results suggested that oxygen availability may affect the competitive capacity of bacteria in endophytic microbiota of black seeds, and selective enriched strains might reduce the mechanical resistance of hard exotesta of black seeds, which contributed to enhance the germinability of black seeds. Mayer and Poljakoo-Mayber  found that one of the possible reasons for the loss of impermeability of seeds was the action of microbes.