Differences in Grain Microstructure and Proteomics of a Broad Bean (Vicia faba L.) Landrace Cixidabaican in China Compared with Lingxiyicun Introduced from Japan

In response to the germplasm resources’ conservation in China, the characters of a superior landrace of broad bean (Vicia faba L.) Cixidabaican (CX) were identified, compared with Lixiyicun (LX) introduced from Japan. The plant morphology and root structure of CX were larger, pods/seeds number and yield per plant were higher, but the size of pods/seeds and single-seed weight were lower than the similar characteristics in LX. The protein content of dry seeds of CX was 4.1% lower than LX, while the amino acids contents showed no difference between the two cultivars. The seed scan electron micrograph showed that the structure of starch granules was similar, while the granules number was lower in CX than LX. iTRAQ-based proteomics showed that 80 differentially abundant proteins (DAPs) were higher, and 45 DAPs were less abundant in the seeds of CX compared to LX, and DAPs were enriched in proteins of carbohydrate and amino acid metabolism. These results verified the importance of the further study of landraces by showing superior traits of CX, which could contribute to the breeding of better-quality varieties.


Introduction
Broad bean (Vicia faba L.), as one of the most important legume crops, is commonly used as vegetables, non-staple food, and stock feed [1,2]. Several studies also showed that broad bean can improve the fertility of soil through coexistence with specific Rhizobium bacteria, and the legume crop residues can be reused for improving soil conditions by incorporating them as green manure [3,4]. It is also a modern nutritious health food and animal feed, as the seeds are a rich source of proteins, mineral components [5,6], carbohydrates [7], and dietary fiber [8]. The seed protein content ranges from 25% to 30% of dry weight, the second highest among legumes after soybean, and a full suite of essential amino acids can be obtained from broad bean protein, except tryptophan and methionine, while lysine content is highest [9]. The content of starch is also high, 37% to 51.5% of dry weight, but all the nutritional contents vary a lot in different varieties, environments, and cultivation conditions [10]. However, despite the many advantages of broad bean, the global acreage of broad bean has decreased from 3.7 to 2.1 million ha since the 1980s, because of the acceleration of urbanization and the deterioration of environments [11,12].
Germplasm resources, as the landraces of a certain region, have been well-adapted to their local environment, and these resources can be used as donors of various important features in well-designed genetic improvement programs for the creation of new varieties [1]. Thus, local landraces, as important vectors of valuable traits and co-adapted genes, should be well-collected and well-conserved [13]. China, as the largest broad bean producing considered to be the most drought-and cold-sensitive growth stage [23], while the evolution of root structure can readily solve this problem [24]. The root architecture of CX was much larger and longer than LX, which increased the water absorption capacity of CX, and hence, decreased the risk of drought effects ( Figure 1C). At the mature stage, the plant phenotype of CX showed a greater plant height, but the fresh pods were significantly smaller than those in LX. The pod length and pod width of CX decreased by 31.1% and 18.2% compared with LX; consequently, the seed size of CX was much smaller than LX. Furthermore, seed pods of CX grew at both the middle and bottom of the plant, while the pods of LX only grew at the bottom, which made the gravitational center of CX higher than LX, and hence increased the risk of lodging ( Figure 1D).

Pods, Seeds, and Yield per Plant of CX were Higher, while the Sizes of Pods and Seeds were Smaller Than LX
Yield is the most important characteristic of germplasm resource evaluation. The factors for the evaluation of yield are seed size and weight, pods per plant, and seed number per pod [25]. According to our research, though the plant phenotype of CX was significantly larger than that of LX, the size of the seeds was smaller, and seed length, seed width, and seed thickness were 11.7%, 15.3%, and 17.1% lower compared with LX on average of the two years of data (Figure 2 and Supplementary Table S2). According to the ANOVA shown in Supplementary Table S2 and Table S3, the two years' field tests showed the same trends between the two varieties: CX recorded significantly higher plant height and seeds per plant, with less single-seed dry weight than that of LX, and there was no significant difference in the stem width between the two varieties and the two years. The seeds per pod and single-seed fresh and dry weight were smaller by 19.5%, 28.9%, and 34.6% respectively, while pods per plant, single-seed pod ratio, pod weight per plant, and seed number per plant were significantly higher in CX compared with LX (133.5%, 52.6%, 16.3%, and 85.6% greater, respectively). Altogether, the seed yield per plant was 32.0% higher in CX than LX (Figure 3 and Supplementary Table S3). As to the year effect, the size of pods and seeds, pods and seeds per plant, and seed yield per plant in the Year 2 were higher than those in the Year 1 experiment. Significant interaction effects of year × variety were only found in the traits of pod length, seed length, and seed width. These results indicate that these traits are susceptible to environmental conditions, especially during the seed developing stage while being affected by varieties.
As the result of high plant growth vigor, CX had significantly more pods and greater pod weight per plant, but most of the pods were single-seed pods, with small seeds and lower seed weight, which are not preferred for human consumption. The consensus among the plant breeders and consumers is that larger seeds contain more food reserves and nutritional components [26], hence CX was gradually replaced by LX, in spite of the seed yield per plant of CX being higher than LX. However, according to Etemadi's report, the currently available varieties showed some drawbacks, such as high seed cost due to  Yield is the most important characteristic of germplasm resource evaluation. The factors for the evaluation of yield are seed size and weight, pods per plant, and seed number per pod [25]. According to our research, though the plant phenotype of CX was significantly larger than that of LX, the size of the seeds was smaller, and seed length, seed width, and seed thickness were 11.7%, 15.3%, and 17.1% lower compared with LX on average of the two years of data ( Figure 2 and Supplementary Table S2). According to the ANOVA shown in Supplementary Tables S2 and S3, the two years' field tests showed the same trends between the two varieties: CX recorded significantly higher plant height and seeds per plant, with less single-seed dry weight than that of LX, and there was no significant difference in the stem width between the two varieties and the two years. The seeds per pod and single-seed fresh and dry weight were smaller by 19.5%, 28.9%, and 34.6% respectively, while pods per plant, single-seed pod ratio, pod weight per plant, and seed number per plant were significantly higher in CX compared with LX (133.5%, 52.6%, 16.3%, and 85.6% greater, respectively). Altogether, the seed yield per plant was 32.0% higher in CX than LX (Figure 3 and Supplementary Table S3). As to the year effect, the size of pods and seeds, pods and seeds per plant, and seed yield per plant in the Year 2 were higher than those in the Year 1 experiment. Significant interaction effects of year × variety were only found in the traits of pod length, seed length, and seed width. These results indicate that these traits are susceptible to environmental conditions, especially during the seed developing stage while being affected by varieties.
As the result of high plant growth vigor, CX had significantly more pods and greater pod weight per plant, but most of the pods were single-seed pods, with small seeds and lower seed weight, which are not preferred for human consumption. The consensus among the plant breeders and consumers is that larger seeds contain more food reserves and nutritional components [26], hence CX was gradually replaced by LX, in spite of the seed yield per plant of CX being higher than LX. However, according to Etemadi's report, the currently available varieties showed some drawbacks, such as high seed cost due to the large seed size and relatively low pod yield [27]. Through our research, we have shown that LX had larger seed size, higher single-seed weight, and more seeds per pod, but pods per plant and seeds per plant were lower due to the characteristic of podding only at the bottom, which finally made the yield of LX lower. Even so, several superior traits were demonstrated in CX, such as more plant growth vigor and higher number of pods and seeds per plant, which provided better basics for CX to gain higher yield, and these can be utilized for future breeding to develop larger seed size with higher yield at the same time.
Plants 2021, 10, x FOR PEER REVIEW 5 of 13 the large seed size and relatively low pod yield [27]. Through our research, we have shown that LX had larger seed size, higher single-seed weight, and more seeds per pod, but pods per plant and seeds per plant were lower due to the characteristic of podding only at the bottom, which finally made the yield of LX lower. Even so, several superior traits were demonstrated in CX, such as more plant growth vigor and higher number of pods and seeds per plant, which provided better basics for CX to gain higher yield, and these can be utilized for future breeding to develop larger seed size with higher yield at the same time.

Amino Acid Contents were High but Showed no Differences between CX and LX
Amino acids are essential nutrients for human beings to maintain normal physiological functions, and both excessive or insufficient intake of essential amino acids will affect human health; hence, the kinds, contents, and proportion of each amino acid are key aspects to evaluate the quality of food. To study the differences of nutritional quality between CX and LX, seed amino acid contents were determined in the Year 1 field experiment (Supplementary Table S4). From the results, methionine and cysteine were the restrictive amino acids, with the contents lower than 100 mg/100 g dry weight. The contents of leucine, lysine, glutamic acid, aspartic acid, and arginine were high, with glutamic acid having the highest content, consistent with the research of Vioque et al. [28]. The proportions of essential amino acids contents vs. total amino acids were 34.7% and 35.5% in CX and LX respectively, which were consistent with the results of Samaei et al. [9] and almost reached the recommended ratio of FAO/WHO/UNU (36%), thus making broad bean a good essential amino acids source for human beings. The amino acids contents of CX were very similar to LX, which showed that CX had as high a nutritional value as the currently popular variety LX.

Amino Acid Contents Were High but Showed No Differences between CX and LX
Amino acids are essential nutrients for human beings to maintain normal physiological functions, and both excessive or insufficient intake of essential amino acids will affect human health; hence, the kinds, contents, and proportion of each amino acid are key aspects to evaluate the quality of food. To study the differences of nutritional quality between CX and LX, seed amino acid contents were determined in the Year 1 field experiment (Supplementary Table S4). From the results, methionine and cysteine were the restrictive amino acids, with the contents lower than 100 mg/100 g dry weight. The contents of leucine, lysine, glutamic acid, aspartic acid, and arginine were high, with glutamic acid having the highest content, consistent with the research of Vioque et al. [28]. The proportions of essential amino acids contents vs. total amino acids were 34.7% and 35.5% in CX and LX respectively, which were consistent with the results of Samaei et al. [9] and almost reached the recommended ratio of FAO/WHO/UNU (36%), thus making broad bean a good essential amino acids source for human beings. The amino acids contents of CX were very similar to LX, which showed that CX had as high a nutritional value as the currently popular variety LX.

Seed Crude Protein Contents and Granules Were Lower in CX Than LX
Broad beans have been an essential staple food as an important dietary protein source, widespread in the Mediterranean area, including continental areas such as Syria, modern Iraq, Iran, and in Northwest India, Pakistan, and Southern China. The high contents of digestible proteins and carbohydrates in their seeds mainly explain their extensive food use [29,30]. As an important source of plant protein and carbohydrates for human daily nutrient intake, the seed protein of broad bean is also a valuable aspect for germplasm resource evaluation [31]. According to our research in the Year 1 field experiment, protein content of the dry seeds in CX was significantly lower than LX, by 4.1% (Figure 2).
The results of the scanning electron microscopy of broad bean cotyledon cross-sections showed that the overall arrangement of tissues was similar in CX and LX, where the shapes of the starch granules were both long elliptical and spheroidal, so we speculated that the granular structure of different faba bean varieties did not contribute to the different taste. However, with regard to starch, the number of granules per unit area was greater in LX than in CX, which might exert a positive influence on superior taste or mouth feel of LX over CX (Figure 4).

Seed Crude Protein Contents and Granules were Lower in CX Than LX
Broad beans have been an essential staple food as an important dietary protein source, widespread in the Mediterranean area, including continental areas such as Syria, modern Iraq, Iran, and in Northwest India, Pakistan, and Southern China. The high contents of digestible proteins and carbohydrates in their seeds mainly explain their extensive food use [29,30]. As an important source of plant protein and carbohydrates for human daily nutrient intake, the seed protein of broad bean is also a valuable aspect for germplasm resource evaluation [31]. According to our research in the Year 1 field experiment, protein content of the dry seeds in CX was significantly lower than LX, by 4.1% (Figure 2).
The results of the scanning electron microscopy of broad bean cotyledon cross-sections showed that the overall arrangement of tissues was similar in CX and LX, where the shapes of the starch granules were both long elliptical and spheroidal, so we speculated that the granular structure of different faba bean varieties did not contribute to the different taste. However, with regard to starch, the number of granules per unit area was greater in LX than in CX, which might exert a positive influence on superior taste or mouth feel of LX over CX (Figure 4).

Identification of Seed Protein Levels and Differentially Abundant Proteins (DAPs) Significantly Enriched in Carbohydrate and Amino Acid Metabolism
The high protein content of broad bean seeds makes these seeds well-adapted to poultry nutrition. Meeting the protein demand of a growing global population is a challenge from both the yield perspective and the quality perspective. While increasing yield potential through breeders' efforts, it is important to ensure the improvement in protein quantity and quality of broad bean as a protein-rich legume seed [29,30]. Thus, the protein contents and composition are also key factors for plant breeding. Hence, proteomic research was used in the first field experiment to compare the differences in proteins in broad bean seeds between CX and LX, using iTRAQ-based proteomics techniques. A total of 521 proteins were identified, and 125 proteins were differentially abundant proteins (DAPs), among which 80 DAPs were significantly upregulated and 45 DAPs were downregulated in CX compared with LX (Table 1). Thirty up-or down-regulated DAPs remained after the fold change, visualized as heat maps, and the molecular weight (MW), amino acids sequence coverage (AASC), and KOG scores were also recorded ( Table 1). To further determine the function of the DAPs, 125 DAPs were classified according to KEGG functional classification, where the top three most enriched functional items were identified as global and overview maps, carbohydrate metabolism, and amino acid metabolism, and the number of up-and down-regulated DAPs are indicated after each item in the

Identification of Seed Protein Levels and Differentially Abundant Proteins (DAPs) Significantly Enriched in Carbohydrate and Amino Acid Metabolism
The high protein content of broad bean seeds makes these seeds well-adapted to poultry nutrition. Meeting the protein demand of a growing global population is a challenge from both the yield perspective and the quality perspective. While increasing yield potential through breeders' efforts, it is important to ensure the improvement in protein quantity and quality of broad bean as a protein-rich legume seed [29,30]. Thus, the protein contents and composition are also key factors for plant breeding. Hence, proteomic research was used in the first field experiment to compare the differences in proteins in broad bean seeds between CX and LX, using iTRAQ-based proteomics techniques. A total of 521 proteins were identified, and 125 proteins were differentially abundant proteins (DAPs), among which 80 DAPs were significantly upregulated and 45 DAPs were downregulated in CX compared with LX (Table 1). Thirty up-or down-regulated DAPs remained after the fold change, visualized as heat maps, and the molecular weight (MW), amino acids sequence coverage (AASC), and KOG scores were also recorded ( Table 1). To further determine the function of the DAPs, 125 DAPs were classified according to KEGG functional classification, where the top three most enriched functional items were identified as global and overview maps, carbohydrate metabolism, and amino acid metabolism, and the number of up-and down-regulated DAPs are indicated after each item in the color bar ( Figure 5). KEGG pathway enrichment results revealed that valine, leucine, and isoleucine degradation, β-alanine metabolism, histidine metabolism, mismatch repair, and propanoate metabolism were significantly enriched DAPs. The interaction of these 5 pathways is shown in Supplementary Figure S1. The fold change was the ratio obtained by comparing the relative protein abundance in CX with LX (CX vs. LX). Fold change ≥ 1.2 are more abundant, between 0.83 < fold change < 1.2 are unchanged, and fold change ≤ 0.83 are less abundant, p-value ≤ 0.05. n represents null. MW, molecular weight; AASC, amino acid sequence coverage; Score, KOG score. Proteins, being the important products of gene expression, are also the reflection of the difference between different germplasm resources [32]. To further identify and evaluate the difference between landrace CX and the excellent imported variety LX, proteomic research of the seeds is urgently needed. As important components of the antioxidant system, the abundance of peroxidase, catalase, and heat shock protein will increase to keep the reactive oxygen species (ROS) in balance when suffering adversity [33]. According to our proteomics results, L-ascorbate peroxidase, 22 kDa heat shock protein, 18.5 kDa heat shock protein, and catalase were 1.53-, 1.52-, 1.45-, and 1.34-fold higher in CX than those in LX, thus ensuring higher stress tolerance, and which may be the reflection of greater environmental adaption ability of the landrace [34].
Enhanced disease susceptibility protein (EDS1) is essential for SA-mediated defenses and is also a major component of SA signaling [35]. The abundance of EDS1 also obtained higher expression in CX, 1.68-fold higher than LX. Thioredoxins are involved a lot in the posttranslational regulation of protein targets in plants, such as seed germination, carbon assimilation, redox signaling, and radical scavenging [36]. The significant high abundance of thioredoxins in CX might increase the ability of seeds in redox modification, hence delaying the senescence and guaranteeing the quality of seeds for a long time.
STICHEL has been reported to play a key role in the regulation of trichome branches in Arabidopsis, which might be directly involved in the formation of branches in the cytoplasm, by mediating the formation of protein complexes, and hence initiating the for- Proteins, being the important products of gene expression, are also the reflection of the difference between different germplasm resources [32]. To further identify and evaluate the difference between landrace CX and the excellent imported variety LX, proteomic research of the seeds is urgently needed. As important components of the antioxidant system, the abundance of peroxidase, catalase, and heat shock protein will increase to keep the reactive oxygen species (ROS) in balance when suffering adversity [33]. According to our proteomics results, L-ascorbate peroxidase, 22 kDa heat shock protein, 18.5 kDa heat shock protein, and catalase were 1.53-, 1.52-, 1.45-, and 1.34-fold higher in CX than those in LX, thus ensuring higher stress tolerance, and which may be the reflection of greater environmental adaption ability of the landrace [34].
Enhanced disease susceptibility protein (EDS1) is essential for SA-mediated defenses and is also a major component of SA signaling [35]. The abundance of EDS1 also obtained higher expression in CX, 1.68-fold higher than LX. Thioredoxins are involved a lot in the posttranslational regulation of protein targets in plants, such as seed germination, carbon assimilation, redox signaling, and radical scavenging [36]. The significant high abundance of thioredoxins in CX might increase the ability of seeds in redox modification, hence delaying the senescence and guaranteeing the quality of seeds for a long time.
STICHEL has been reported to play a key role in the regulation of trichome branches in Arabidopsis, which might be directly involved in the formation of branches in the cytoplasm, by mediating the formation of protein complexes, and hence initiating the formation of branches. Trichomes, as the mechanical barrier between plants and the environment, is the first defense of plants to adversity [37]. According to our research, protein STICHEL obtained the highest upregulation in CX compared to LX, which might reflect that CX has high potential to grow more trichome branches than LX, hence increasing the environmental adaptive ability of CX.
In contrast, oleosin protein, histone, and glycinin, which were the important kinds of broad bean proteins, were significantly downregulated in CX compared with LX (Table 1), with the same trends as the seed crude protein contents measured before, which were also decreased in CX compared to LX.
Due to the increasing use of plant proteins from broad beans, a few challenges have appeared, such as the formation of grainy or beany off-flavors [38]. Broad beans are known to have high levels of lipoxygenase (LOX), which acts as the key enzyme of the lipid-degrading LOX pathway [19], with the main results of the LOX pathway being the generation of numerous volatile lipid oxidation products [39]. Great efforts have been undertaken to control the activity of LOX, and hence solve the off-flavor problem [40]. From our results, lipoxygenase in CX was significantly lower than in LX, which might be a good resource for the future study on how to eliminate the beany off-flavors.

Experiment Design, Plant Materials, and Harvest
The field trial was conducted in Cixi, Zhejiang province, China (30. In each plot, 10 plants were randomly selected for pod harvest and yield determination. Pods per plant, seeds per pod and per plant, single-seed pod ratio, pod weight per plant, seed fresh and dry weight, and seed yield were recorded in the mature harvest period.

Determination of Seed Protein and Amino Acid Contents
Seed protein and amino acid contents were measured based on the first field experiment for the following ultrastructure observation and proteomics analysis. Dry and healthy seeds were selected for protein content measurement. After removing the seed coat carefully, the seeds were placed in a whirlwind pulverizer FG-11 and ground into powder, and then sealed tightly for future use. A total of 1 g of broad bean powder was weighed for crude protein content measurement, according to the Kjeldahl method (AOAC, 2006). A total of 0.2 g of powder was weighed for amino acid content determination using an AAA-400 amino acid analyzer according to Lisiewska [8]. The proportion of essential amino acids to total amino acids was determined as follows: E/T% = (IlE + Leu + Lys + Met + Phe + Thr + Val + Try)/ (IlE + Leu + Lys + Met + Phe + Thr + Val + Try + Ala + Arg + Asp + Cys + Glu + Gly + His + Pro + Ser + Tyr) × 100%. (1)

Ultrastructure Observation of Broad Bean Cotyledon Cross-Sections
The ultrastructure of broad bean cotyledon cross-sections was observed through scanning electron microscopy according to Chen et al. [41], with minor modifications. In brief, the seed coat was carefully removed, and the cotyledons were cut to obtain a slice with natural fracture surfaces remaining. The fracture surfaces were sputter-coated with gold and palladium, and the ultrastructure of the natural fracture surface of each variety was observed using a GeminiSEM 300 (Oberkochen, Germany). To compare the differences in ultrastructure between CX and LX, 100-, 500-, and 2000-fold magnifications were used, especially for the structure and arrangement of starch granules.

Proteomics Analysis
The fresh broad bean seeds of CX and LX were harvested and sampled at the mature stage in the Year 2018-2019, and a total of 3 replicates were used. A subsample of 0.8 g of seeds was taken for the preparation of protein, and for the quantification, concentration detection and digestion, iTRAQ-labeling and peptide fractionation, HPLC and mass spectrom-eter detection were conducted according to Zeng et al. [42]. The proteins were identified via NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 29 May 2019)), where the taxonomy search was for Vicia faba, on 28 May 2019. The raw MS/MS data were searched using Mascot version 2.3.02 against the selected database, and the variable modifications of amino acids were oxidation and iTRAQ8plex and the fixed modifications were carbamidomethyl and iTRAQ8plex. The overview of protein identification is shown in Supplementary Table  S5. Bioinformatics analysis of the proteomic data was as described by Zeng et al. [42]. The quantitative ratios (CX/LX) of the identified proteins greater than 1.2 were considered as upregulated (p ≤ 0.05), while quantitative ratios less than 0.83 were considered as downregulated (p ≤ 0.05). The mass spectrometry proteomics data have been deposited to the Pro-teomeXchange Consortium (http://proteomecentral.proteomexchange.org (accessed on 29 May 2019)) via the iProX partner repository, with the dataset identifier PXD026415 [43].

Statistical Analysis
The data were presented as mean values of each treatment. Statistical analysis was performed using Data Processing System (DPS) statistical software. Student's t-tests were performed to evaluate significant variety differences between CX and LX at the significance level of p ≤ 0.05 [44]. Origin version 7.5 was used for the preparation of graphs.

Conclusions
Increasing urbanization and environmental change has led to decreases in cultivation and the disappearance of several conventional local genetic resources, thus making it imperative that germplasm resources are conserved as a source for plant breeding [45]. China, as the country with the largest cultivation of broad beans and the second center of broad bean genetic resources, has lots of excellent landraces and traits for breeders to use to improve currently popular varieties; hence, there is an urgent need to evaluate the landraces of China. In this paper, the morphology, yield, protein contents, amino acid contents, starch contents, and proteomes of landrace Cixidabaican (CX) were identified and compared with Lingxiyicun (LX), which was introduced from Japan. In summary: (1) CX grew taller than LX, but the seed size of CX was smaller than LX, and seed weight was lower. (2) Seed yield of CX was higher due to a greater number of pods, but the protein content was lower than LX. (3) Proteomics analysis showed that, as a result of long-term adaption to the local environment, heat shock proteins, L-ascorbate peroxidase, catalase, EDS1 protein, thioredoxins, and STICHEL protein were upregulated, which could indicate increased environmental adaption ability. (4) Downregulated LOX activity in CX can also be a useful trait for future work on the alleviation of off-flavors.
In conclusion, although CX has been replaced by LX nowadays, this landrace still possesses many excellent traits, which may be important for future breeding improvements. For example, because of the long-term adaption to the local environment, environmental responsive proteins were upregulated in CX, and these proteins and related genes can be utilized for breeding development through genetic engineering to endow the varieties introduced from foreign areas with a higher ability for environmental adaption. Cultivation techniques, such as hybridization, backcross techniques, and population design in open pollinated conditions, are also expected to increase genetic gains, and hence combine beneficial characteristics (e.g., higher yield and lower off-flavors) of CX with LX, and consequently, develop tasteful varieties with higher yield.