Evaluation of Fine Root Morphology and Rhizosphere Environmental Characteristics of the Dioecious Idesia polycarpa Maxim
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1. Introduction
Due to the long-term evolution of dioecious plants, there are gender differences in morphological characteristics, resource utilization, and biomass accumulation among individuals. This difference is generally related to different reproductive costs [1]. Roots, as one of the most important underground organs of plants, can not only fix the surface but also have important functions in absorbing, converting, and storing [2]. Studies have found differences in root morphology between male and female plants, showing gender dimorphism. The root growth rate and quality of Chaenomelis fructus female plants are significantly higher than those of male plants [3]. In the case of sufficient water, the Populus cathayana female plants have strong competitive ability, but under drought stress, the male plants have strong competitive ability [4]. The intensity of leaf litter affected the reserves of thick and fine roots [5]. The total length, surface area, and volume of female P. cathayana roots of heterosexual adjacent plants are significantly higher than the male plants [6]. Moreover, fine roots are the most active part of plant roots [7] and are the main bearers of nutrients that plants absorb. The growth of the plants is mostly determined by fine root functional traits, including root length, root diameter, specific root length, and root tissue density [8]. Exploring changes in plant root growth helps in understanding plant growth patterns.
The rhizosphere is crucial for exchanging materials and energy between plants and the external environment. Nutrient elements such as N, P, and K in rhizosphere soil are the basis for plant growth and development, but nutrient utilization by male and female plants is quite different due to gender dimorphism [9]. Under full light conditions, C/N and C/P in the coarse roots of female Pinus yunnanensis increased while decreasing in the coarse roots of male plants [10]. Song et al. [11] showed that female P. yunnanensis needed more nitrogen to maintain growth and reproduction. Fu et al. [12] found that the contents of total nitrogen (TN), total phosphorus (TP), total potassium (TK), available phosphorus (AP), and AK in the rhizosphere soil of male plants were significantly higher than those of female plants. Studies have shown that some nutrients in the soil need to be transformed by microorganisms to be absorbed by plants [13,14]. Rhizosphere microorganisms are nutrient cycle catalysts between plant roots and rhizosphere soil [15]. Zhou et al. [16] showed that host genotype affected the composition of plant rhizosphere microorganisms. The relative abundance of Planctomycetaceae, Xanthomonadaceae, and Cytophagaceae in the rhizosphere soil of male Populus euphratica plants was higher than that of female plants [17]. In the interaction between plants and rhizosphere microorganisms, plants affect the composition of soil microbial communities through root exudates, such as sugars, secondary metabolites, and organic acids, and accelerate nutrient decomposition through the action of microorganisms [18,19]. Du et al. [20] found that the total amino acid content in the rhizosphere soil of Beta vulgaris increased with B. vulgaris growth. Mille et al. [21] showed that plant genotypes influenced differences in metabolic profiles in rhizosphere soil. Xia et al. [22] found that the acid phosphatase content in rhizosphere soil in male poplar was higher than that in females. Plant characteristics determine the interaction of plant–soil–microorganisms. Exploring the root environmental differences between male and female plants helps us understand plant biological characteristics.
However, Idesia polycarpa is a dioecious plant of the Idesia genus in Salicaceae. Idesia polycarpa plays a crucial role in landscaping and greening efforts, and their attractive shapes and lush flowers contribute to the aesthetic appeal of outdoor spaces [23]. The fruit yield is high, the oil content is as high as 35%, and the oil is rich in nutrients, such as linoleic acid, vitamin E, and sterols. It is a “double high” vegetable oil with high nutrition and value [24]. At present, relevant scholars have studied the growth of I. polycarpa from the above-ground parts of seeds [25], leaves [26], and branches [27], and there are few reports on its underground parts ([28], unpublished data). Our study used the dioecious Idesia polycarpa Maxim species to explore the differences in fine root morphology, rhizosphere soil nutrients, and their metabolites in male and female plants. This study aimed to gain a deeper understanding of the biological properties, provide a theoretical basis, and lay a foundation for field management of Idesia polycarpa.
2. Materials and Methods
2.1. Overview of the Test Area
The experimental area is located at the Forestry Experimental Station of Henan Agricultural University in Zhengzhou City, Henan Province (112° 42′ 114° 14′ E, and 34° 16′ 34° 58′ N) (Figure 1). The mean annual temperature of this site is 14.2 °C, the annual precipitation is 623.30 mm, and the frost-free period of the year is 220 days. Soils in Zhengzhou City are characterized by low organic matter content, high effective phosphorus content, and coarse texture [29]. The soil texture of this trial site is sandy loam.
2.2. Experimental Materials
In December 2020, three male and three female plants of 7-year-old I. polycarpa, with the same growth and no obvious pests and diseases, were selected from the experimental site. These plants were part of a plantation with a spacing of 1.5 m × 1.5 m. According to the method introduced by Johnson et al. [30], six plants were selected at the experimental site, two minirhizotron tubes were placed in the north–south direction of each experimental material at a 45° angle to the ground, and a total of 12 minirhizotron tubes were embedded. The minirhizotron tubes (length: 100 cm; outer diameter: 7.0 cm; inner diameter: 6.40 cm) had a 45° angle with the ground, about 20 cm was exposed to the ground, and the vertical observation distance was about 40 cm. The leaky part of the micro root canal was wrapped in black tape, and a layer of aluminum foil was added. During the test, the micro root canal was protected against interference [31,32]. Roots were scanned and observed using the Plant Root Growth Monitoring System CL-600 (CID, Camas, WA, USA) during the 25th day of May 2021 (D1; flowering stage, female: CX5; male: XS5), July 2021 (D2; fruit accumulation stage), and October 2021 (D3; fruit maturity stage, female: CX10; male: XS10). The fine root samples were collected at all stages (D1, D2, and D3) and treatments (female and male), and the soil was cleaned and removed to keep the root system intact. Subsequently, the roots were then cut and spread flat in a root tray with distilled water. The water depth was required for the roots to be completely covered, a glass rod was used to place them in a stretched state, and they were finally covered with a backplate for root scanning. The image acquisition area was 21.59 cm × 19.57 cm. At the same time, the sterilized root drill was used to drill 0–20 cm soil samples in the four directions of the selected plant, and the distance was 20 cm from the tree trunk [33]. After mixing evenly, the samples were placed in an incubator with dry ice and returned quickly to the laboratory. Subsequently, one part of the soil sample was placed in a refrigerator at −80 °C to determine soil microorganisms and metabolites, and the other part was dried in the laboratory to determine soil nutrient content.
2.3. Fine Root Morphology Measurement
WinRHIZO Tron MF (Regent Instruments, Inc., Québec, QC, Canada) root analysis software was used to analyze photos of fine roots (the diameter between 2 and 5 mm) taken in each period, and the total root length, root tip number, average root diameter, and total root surface area of the fine roots of male and female I. polycarpa in each stage were obtained. The IBM SPSS Statistics v. 26 (IBM Corp., Armonk, NY, USA; available online: https://www.ibm.com, accessed on 19 January 2024) program was used for ANOVN data analysis, and Origin 23b (available online: https://www.originlab.com, accessed on 19 January 2024) was used to draw bar charts.
2.4. Soil Nutrient Determination and Analysis
According to the soil agrochemical analysis [34], the soil pH value was determined with the pH meter method with a water–soil ratio of 2.5:1. An automatic elemental analyzer (EURO EA3000) (EuroVector S.p.A., Leuven, Belgium) was used to determine the TC and TN. The AN was determined by 1 mol/L NaOH extraction-acid titration. The AP was determined using the 0.5 mol/L NaHCO3 extraction-molybdenum antimony colorimetric method. The AK was determined using a 1 mol/L CH3COONH4 extraction flame photometer.
2.5. Genome Sequencing
Total DNA was extracted as instructed in an E.Z.N.A. ® soil kit (Omega Bio-tek, Norcross, GA, USA). The concentration and purity of DNA were measured using NanoDrop2000, and DNA quality was measured using 1% agarose gel electrophoresis. Primers 341F (5′-ACTCCTACGGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3-V4 region of the bacterial 16 S r RNA gene. The ITS1-ITS2 mushroom region was amplified with ITS1-1F-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS1-1F-R (5′-GCTGCGTTCTTCATCGATGC-3′) by PCR. PCR reaction conditions: 3 min before denaturation at 95 °C, 27 cycles (95 °C and 30 s, 55 °C annealing 30 s, 72 °C extension 30 s), 72 °C extension 10 min to purification of amplified products and construction of library. Once the library was qualified, it was sequenced with the Miseq PE300 platform of Illumina. The Wekemo Tech Group Co., Ltd., Shenzhen, China, was commissioned for DNA extraction, PCR amplification, and sequencing.
2.6. Bioinformatics Analysis
Quality control and de-noising were performed on the sequencing results with the QIIME2 plugin. Based on similarity, the sequence was grouped into the Operational Taxonomic Unit (OTU), and the species were annotated. The Alpha diversity index was computed using QIIME2 (Quantitative Insights Into Microbial Ecology vs. 2022.11) software [35]. R software (V4.1.0) was used to analyze the difference in Alpha diversity index between groups, and a Venn diagram, species relative abundance diagram, microbial and fine root trait redundancy analysis (RDA) diagram, and association heat maps were drawn.
2.7. Determination of Rhizosphere Soil Metabolites
The samples were vacuum freeze-dried and grounded (30 Hz; 30 s) to powder with a grinding instrument. The 0.5 g sample was weighed, and 1 mL methanol:isopropanol:water (3:3:2, V:V:V) extract was added. After shaking at room temperature for 3 min, the sample was placed in an ice water bath for 20 min. After centrifugation at 12,000 r/min for 3 min at 4 °C, the supernatant was transferred to the injection bottle. The 0.020 mL internal standard (10 μg/mL) was added, dried with the nitrogen-blowing instrument, and placed in a freeze-drying machine for freeze-drying. Subsequently, 0.1 mL of methoxyamine pyridine (0.015 g/mL) was added and placed in an oven at 37 °C for 2 h, followed by 0.1 mL of BSTFA (with 1% TMCS) in an oven at 37 °C for 30 min to obtain a derivatization solution. The derivatization solution was diluted to 1 mL, filtered by a 0.22 μm organic phase needle filter, stored in a refrigerator at −20 °C, and injected for GC–MS detection.
This study detected the metabolites using gas chromatography (Agilent8890-5977B, Santa Clara, CA, USA). The qualitative and quantitative analysis of metabolite data was performed based on Maiwei’s self-built S_TMS_MWGC database.
2.8. Metabolite Data Analysis
Using R software, the metabolism of different samples was compared and analyzed, and the results were analyzed using multivariate statistics. Based on the variable importance projection (VIP), the results obtained by OPLS-DA, VIP ≥ 1, and p < 0.05 were defined as significantly altered metabolites (SCMs), and the corresponding differential metabolites were submitted to the KEGG (Kyoto Encyclopedia of Genes and Genomes) website to obtain metabolic pathways. Using R software (V4.1.0), the redundant analysis (RDA) and relative heat maps of significant difference metabolites and fine root characters were plotted.
4. Discussion
The plasticity expression of plant traits plays an important role in plant adaptation to the environment [36]. As the plant’s most active and sensitive component, the fine roots can absorb nutrients by expanding soil space through proliferation and growth [37,38]. They can also adapt more efficiently to the soil environment by changing their functional characteristics, such as fine root morphology and configuration characteristics, to optimize resource acquisition efficiency [39]. Female plants require more reproductive growth than male plants, so they differ in resource demand and distribution [40,41]. In this study, the total root length, total root surface area, total root volume, root tip number, and average root diameter of the fine roots of female plants were greater than those of male plants, indicating that female plants had stronger underground competitiveness than male plants. Studies have shown that the extension length and diameter of new branches of female plants are higher than those of male plants [42,43], indicating that female plants have more developed roots and absorb more nutrients, which is similar to the results of this paper. The growth of plants is greatly affected by the season, and spring yields the maximum growth. In this study, the fine roots of male and female plants were not significantly different in the flowering stage (May). With time, the fine root biomass of female plants increased rapidly, and the female plants’ growth rate was greater than that of male plants. The difference reached its maximum at the fruit accumulation stage (July), similar to the results of Zhao et al. [44]. Appropriate climatic conditions and their increased soil nutrient demand promoted the rapid growth of fine roots of I. polycarpa.
Soil microorganisms play an important role in the rhizosphere microecological environment of plants. They are the driving force for transforming available soil nutrients and the source and reservoir of available soil nutrients. The rhizosphere microbial community structure of plants has strong habitat specificity [45]. Specific plants have unique rhizosphere microbial communities [46]. In this study, it was found that the diversity of bacteria and fungi in the rhizosphere of male and female plants was similar, and the dominant bacteria in the rhizosphere soil of male and female plants were Proteobacteria, Acidobacteria, Actinobacteria, Ascomycota, and Mortierellomycotabe. It shows that the composition of bacterial and fungal communities in the rhizosphere of male and female plants is relatively fixed. Guo et al. [47] showed gender-specific bacterial and fungal communities in the rhizosphere soil of male and female P. euphratica in natural forests, but no specific colonies were found in the rhizosphere in our present study. It may be that the ecological conditions are relatively simple under plantation land conditions. However, in this paper, the relative abundance of bacteria such as Bacillus, Paenibacillus, and Arthrobacter and fungi such as Chaetomium, Mortierella, and Volutella in the rhizosphere soil of males and females differed during the flowering and fruit ripening stages. The female plants need to invest more in reproductive costs than males, and root transfer will show more complex root nutrient exchange. The roots promote the proliferation and extinction of specific microorganisms and improve the rhizosphere environment by shaping the physical and chemical environment of the special rhizosphere soil [48].
Rhizosphere soil metabolites are a comprehensive reflection of root–soil–microbial activities [49]. As a signal substance and microbial nutrient source, they regulate the structure and diversity of rhizosphere microorganisms and plant growth and development through microbial activities. It is an important part of the rhizosphere microecosystem [50]. Our study found that the significant differential metabolites in the rhizosphere soil of male and female plants during the flowering stage were greater than those at the fruit ripening stage. Both male and female flowering plants also achieved vegetative and reproductive growth, increased nutrient demand, and had a strong exchange of roots and soil matter. Further analysis showed that significantly different metabolites between male and female plants were more carbohydrates. Li et al. [51] found that amino acids, sugars, sugar alcohols, and other substances in the soil can provide certain nutrient support for the growth of American ginseng from late autumn to early spring, and sugars are also the main carbon source of microorganisms [52]. The rhizosphere of female plants accumulates more carbohydrates, which can increase the richness of rhizosphere microorganisms and provide a nutrient guarantee for their growth and development. Arabinofuranose, Glucose 1, and D-Allose 2 showed significant differences in the flowering and fruit ripening stages of I. polycarpa, and their contents, which may be closely related to the root activity of I. polycarpa. The KEGG metabolic pathway differential enrichment analysis found that more carbohydrate synthesis and metabolic pathways were enriched in the rhizosphere soil of male and female plants at the flowering stage, consistent with the expression of significantly different metabolites in this period. More stress-resistant material synthesis and metabolic pathways and phosphorus metabolic pathways were enriched during fruit material accumulation. During the fruit material accumulation period, I. polycarpa mainly carried out reproductive growth, and more nutrients were distributed to the fruit [53]. The trees’ resistance decreased, and the synthesis and metabolism of stress-resistant substances in the rhizosphere soil were more conducive to improving plant resistance and vitality.
Plant roots are affected by rhizosphere environmental factors while creating a unique environment. Rhizosphere nutrients are substances that can be used directly by roots and are closely related to root growth. In this paper, RDA analysis showed that AK, Pedomicrobium, Chaetomium, and Glucose 1 in rhizosphere soil had the greatest influence on fine root traits. Potassium can control IAA oxidase activity, increase IAA content, promote carbohydrate transport to roots, and promote root growth [54]. However, correlation analysis showed that the AK content in the rhizosphere soil of I. polycarpa negatively correlated with fine root traits. Chen [55] confirmed that applying potassium fertilizer based on nitrogen and phosphorus can significantly affect root morphology and growth. Our study indicated that a reasonable nitrogen, phosphorus, and potassium ratio can promote root development. This may be one reason for the negative correlation between fine root traits of I. polycarpa and AK content in the rhizosphere. In a later study, the nutrient distribution ratio can be tested to find the optimal soil nutrient match for the growth of I. polycarpa fine roots. Pedomicrobium and Chaetomium are important in removing metal ion pollution from the environment, degrading organic matter in the soil, inhibiting pathogens’ growth, and indirectly affecting roots by regulating environmental conditions [56]. However, both had a significant negative correlation with fine root traits, indicating that both microorganisms strongly inhibited fine root growth. Among other rhizosphere microorganisms, most are positively correlated with the fine root traits of I. polycarpa, which is similar to the results of previous studies on this plant [57]. The interaction between plants and rhizosphere microorganisms is mutually beneficial, and more secondary metabolites will accumulate in rhizosphere soil when the two interact. As the main carbon source of microorganisms, Glucose 1 can be widely used by many microbial groups [58], which is conducive to the growth of rhizosphere microorganisms. Rich microbial diversity can promote the growth and development of plant roots [59]. In this study, metabolites in the rhizosphere of I. polycarpa were found to have more negative effects on the fine roots of I. polycarpa. Organic matter in plant rhizosphere soil has been found to have toxic effects on plants [60]. For example, Marmesin, Cytisine, and Indole-3-acetic acid are in the rhizosphere of continuous cropping. American ginseng had varying degrees of inhibition on the growth of American ginseng roots [61]. Allelochemicals may also be present in the rhizosphere metabolites of I. polycarpa, requiring further investigation.
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