Exploration of Biomass-related Genes in the FOX-superroot Lines of Lotus corniculatus and its Impact on Agronomic Traits

Birdsfoot trefoil (Lotus corniculatus L.) native to Europe is a widely used perennial forage legume crop globally (Steiner and de los Santos, 2001). It is known for its cross-pollination characteristics and tetraploid nature (2n = 4x = 24) (Grant and Small, 1996). It belongs to the same genus as the model plant L. japonicus, a diploid species for which a complete genome sequence is readily accessible (Li et al., 2020). Super-growing roots (SR) is a first-growing root culture found in L. corniculatus that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions (Akashi et al., 1998, 2003). This plant material has sustained its characteristics for 25 years after isolation and also allows for protoplast culture (Akashi et al., 2000) and genetic transformation (Tanaka et al., 2008), which makes it a useful research material for the analysis of root physiology and gene function.
       
As the world population grows, it becomes essential to improve crop production and efficiency. To meet this demand, agriculture must increase production through conventional or genetic modification breeding technique. Genetic transformation as a powerful tool that can answer this problem offers the opportunity to improve and modify plants for useful traits. In parallel, the sequencing of entire genomes for many plant species (Kersey, 2019; Sugumar et al., 2024), has provided a wealth of genetic information, yet the functions of many genes remain unclear. This  underscores the need to identify and understand agronomically useful genes to fully harness the potential of genetic transformation.
       
Full-length cDNA Over-eXpressor gene-hunting system (FOX gene-hunting system) enables comprehensive   analysis of gene function in plants by ectopically overexpressing full-length cDNA. This system has been developed in Arabidopsis thaliana (Ichikawa et al., 2006) and rice (Nakamura et al., 2007), which were limited to model plants. SR unique culture system was applied to develop a FOX line with Arabidopsis cDNA introduced into L. corniculatus (Himuro et al., 2011). Therefore, this FOX line has made it possible to explore agronomic useful genes in legume crops.
       
In this study, we explored biomass-related genes by evaluating 66 FSLs integrated with Arabidopsis cDNA. Specifically, two FSLs with distinctive characteristics were grown in the greenhouse until the flowering stage and their agronomic traits were investigated in detail. This study represents the first attempt to utilize the FOX hunting system to search for agriculturally useful genes in legume crops.

Plant materials and culture conditions
 
The plant material was 66 FOX superroot lines (FSLs), which were previously generated by introducing the Arabidopsis-FOX Agrobacterium library (Ichikawa et al., 2006) into bird’s-foot trefoil (Lotus corniculatus) cv. Viking (Himuro et al., 2011). These FSLs were maintained in half-strength Murashige and Skoog (1/2 MS) medium without hormones as described by Murashige and Skoog (1962), solidified with 0.7% (w/v) agar and grown in a growth chamber with a 16/8 photoperiod. The experiment was conducted from 2014 to 2016 at the Frontier Science Research Center, University of Miyazaki, Japan.
 
Measurement of root length and plant length in FOX superroot lines
 
Sixty-six FSLs maintained in 1/2 MS solid medium were cut at the internodes of the third to fourth expanded leaves and transferred to fresh 1/2 MS solid medium. Two weeks later, they were transplanted into 6.5 cm² pot filled with vermiculite. Following four weeks of cultivation in a growth chamber with a 16/8 photoperiod, the plant length and root length was measured with three replications on each line. Total root length was measured by image analysis using WinRHIZO™ Arabidopsis software (Regent Instruments Inc., Canada).
 
Confirmation of CPD (FSL#83) and SYNC1 (FSL#121) genes expression by reverse Transcriptase-polymerase chain reaction (RT-PCR)
 
To further examine the agronomic traits of distinctive morphology FSL#83 and FSL#121, the expression of their transgenes, CPD and SYNC1, respectively was confirmed by RT-PCR. The extraction of total RNA from leaf, stem and root tissues followed the TRIzol Reagent (Invitrogen) protocol. Complementary cDNAs were synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany). RT-PCR was conducted with gene-specific primers, including CPD primers: 5'-TCCTCCTCCTCTCTTCCATCGCCG-3' and 5'-ACGGCGCTTCACGAAGATCGGGTA-3' and SYNC1 primers: 5'-CGTAGATAACGAACAATGGCTGATG-3' and 5’- GATCTCTTCCGGATTTTACCCAAC-3'. Internal control for normalization utilized a primer pair amplifying cDNAs for ACTIN1: 5'-ACAATGAGTTGCGTGTTGCT-3' and 5'-ACTCA CACCATCACCGGAAT-3'. The RT-PCR was performed by preheating 94°C for 5 min and followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 1 min. PCR products were separated by 2.0% agarose gel electrophoresis.
 
Measurement of morphological traits in FSL #83 and #121
 
The FSL#83 and #121 were transferred to a 1/5000a pot filled with sterile soil (Miyazaki Shodo) and grown until the flowering stage in the greenhouse of University of Miyazaki, Japan for another four months. Morphological traits such as stem diameter, internode length, leaf length and width and stem, branches, node and flower number were measured in the fourth month of cultivation. Additionally, dry matter weight was measured after harvest as summarized in Table 1. The data were analyzed using one-way analysis of variance (ANOVA) to determine significant differences among the three plant lines for each of the 13 measured traits with five replications. Following significant ANOVA results, Tukey’s Honestly Significant Difference (HSD) post-hoc test was conducted to identify specific group differences.
 

Phenotypic variations and selection of high biomass lines in FSLs
 
In this study, we analyze the functionality of Arabidopsis cDNA in SR using the FOX hunting system, with a focus on biomass and demonstrated its usefulness in legume crops. Sixty-six lines were used to measure plant length and total root length as biomass traits in early plant growth stage. The trait values were widely distributed around the SR and their morphology was affected by the introduced Arabidopsis cDNA (Fig 1). The non-transgenic SR used as a control had a plant length of 25.8 cm and a total root length of 63.1 cm. Among the tested lines, there were 18 lines displayed greater plant length and 48 lines exhibited greater total root length than SR. To identify lines with superior biomass, a threshold of 30 cm in plant length and 100 cm in total root length was set. Fifteen lines surpassed these criteria were selected and their respective transgenes were listed in Table 2.
 

 

       
The initial stages of plant growth are influenced by both above-ground and under-ground development, making it a crucial factor in biomass production (Berhongaray et al., 2013; Singh et al., 2016). Identifying the genetic factors that control plant growth is essential for developing crops with improved yields. Three FSLs (#110, #121 and #125) surpassed the threshold of 30 cm in plant length. Their transgenes were Arabidopsis Selenoprotein.Rdx type: At5g58640 (FSL#110), SYNC1 gene: At5g56680 (FSL#121) and Serine/arginine repetitive matrix-like protein: At4g22190 (FSL#125). Selenoprotein Rdx belongs to the thioredoxin-like family and plays a critical role in various aspects of plant biology, such as photosynthesis, metabolism, growth, development and responses to environmental stresses (Dikiy et al., 2007; Chauhan et al., 2017). The introduction of serine/arginine proteins in FSL#125 not only contribute to increased plant length but also to longer root development. The SYNC1 gene is responsible for encoding an asparaginyl-tRNA synthetase, which catalyzes the synthesis of key amino acids in seeds, influencing plant morphology and resulting in a significant increase in plant length, number of branches and number of branch nodes, all of which are yield components of soybean (Arifin et al., 2019). Serine/arginine proteins are vital splicing factors that play crucial roles in spliceosome assembly and splicing regulation. Loss of function of these proteins can lead to various changes in plant morphology and development, including serrated leaves, late flowering, shorter roots and abnormal silique phyllotaxy (Yan et al., 2017).
       
Over half of the lines displayed total root lengths higher than SR, with 13 FSLs surpassing the 100 cm threshold (Fig 1). These 13 FSLs with superior root growth (#6, #35, #41, #45, #55, #77, #83, #95, #98, #102, #124, #125 and #126) were each introduced with specific Arabidopsis cDNA with various plant functions, such as membrane transport (#35), growth and development (#41, #55), viral replication (#45), hormone biosynthesis (#83), redox regulation (#95), metabolic pathways (#98), photosynthesis (#102), calcium signaling (#124) and Zinc Finger Protein associated with transcription factors (#126). For instance, FSL#35 exhibited the highest total root length of 134 cm and expressed the outer envelope pore 24B-like protein: At5g42960, essential for transporting nutrients like sugars, ATP and amino acids crucial for plant growth (Zybailov et al., 2008). Similarly, the expression of Cytokinin-Responsive Growth Regulator influenced root development in FSL#55, as cytokinins regulate processes such as cell division and root regeneration (Higuchi et al., 2004; Marhavý et al., 2011). Moreover, the extensive root length in FSL#95 may be attributed to redox regulation induced by Thioredoxin 3: At5g42980, which participating in thiol-disulfide exchange reactions and contributing to the maintenance of the redox homeostasis necessary for various cellular processes in plants (Gelhaye et al., 2005). Understanding the role of these genes related to biomas production in L. corniculatus can be useful for improving pasture productivity. Furthermore, we expect that this basic research will lead to applications in breeding other legume crops.
 
Transgene expression in FSLs #83 and #121
 
Among the 15 superior FOX superroot lines (FSLs), #83 integrated with the Constitutive Photomorphogenic Dwarf (CPD) gene: At5g05690 demonstrated longer total root length and plant length similar to SR, while also displaying an increased number of side branches. Similarly, #121 carrying the Asparaginyl-tRNA synthetase (SYNC1) gene: At5g56680, exhibited a higher plant length and thicker stems. To further examine the agronomic traits of distinctive morphology FSL#83 and FSL#121, the expression of their transgenes in the leaf, stem and root was initially confirmed by RT-PCR. Individual gene-specific primers were used to amplify each transgene, with ACTIN1 serving as an internal control. The RT-PCR products were then separated in a 2.0% agarose gel, using the SR line as a control in each experiment. The results revealed that under the regulation of the CaMV35S promoter, CPD and SYNC1 were overexpressed constantly in all tissues of FSL #83 (Fig 2a) and #121 (Fig 2b), respectively.
 

 
The effect of CPD and SYNC1 gene overexpression on morphological traits in FSLs #83 and #121
 
SR, FSL#83 and FSL#121 were cultivated for 4 months until they reached maturity to evaluate the influence of overexpressing the CPD and SYNC1 genes on agronomic traits. The results were summarized in Table 3 and Fig 3, where it was observed that FSL#83 showed higher internode length, number of stems, number of flowers and above-ground dry matter weight compared to SR. The CPD gene encodes the cytochrome P450 enzyme CYP90A1, which roles an important process in the brassinosteroid biosynthesis pathway. Brassinosteroids play crucial roles in various physiological and developmental processes in plants, including cell expansion, cell division, vascular development and stress responses (Sui et al., 2012; Godara et al., 2017). Our results showed that the increase in number of flowers and above-ground dry matter weight in FSL #83 as a result of CPD expression, aligns with previous research on the overexpression of brassinosteroid synthesis genes in Arabidopsis (Choe et al., 2001), pear (Zheng et al., 2020), rice and maize (Sun et al., 2021). FSL#121 exhibited higher plant lengths and number of stem nodes compared to SR. SYNC1, the Asparaginyl-tRNA synthetase gene, is crucial for transferring amino acids to cognate tRNA molecules, essential for accurate protein translation (Friedman et al., 2019). Higher expression levels of the SYNC1 gene enhance the activity of enzymes involved in the synthesis of amino acids such as asparagine, lysine, aspartic acid, alanine and histidine, leading to improved plant growth, including increased plant height, branching, stem thickness and fresh weight of shoots in transgenic soybean (Arifin et al., 2019). In this study, the exploration of biomass-related genes using the L. corniculatus FOX Line is expected to be applied to the same legume crops. Transformation of the SYNC1 gene into soybean resulted in the plant growing to twice the height compared to the wild type, along with an increased number of branches, which aligns with the findings of this study (Arifin et al., 2019). Next, we would like to create transgenic soybean with overexpressed CPD gene and determin its effect of increased number of flowers on the yield.
 

 

Identifying genes associated with biomass is crucial for meeting the growing demand for food. In our study, we explored these genes by studying 66 FOX superroot lines (FSLs) that integrated with Arabidopsis cDNA. We used the FOX hunting system to analyze the functionality of Arabidopsis cDNA in FSLs, focusing on biomass traits and demonstrated its potential in legume crops. The 66 FSLs showed diverse plant heights and root lengths during the early growth stage, with some lines exhibiting superior growth. Fifteen FSLs were identified with superior growth characteristics, surpassing thresholds of 30 cm for plant length and 100 cm for root length. These superior FSLs were each integrated with Arabidopsis cDNA, categorized into genes related to various functions such as membrane transport, photosystems, phytohormone synthesis, amino acid synthesis and two unknown genes. Two FSLs with distinct characteristics were grown for 4 months in a greenhouse until the flowering stage and their agronomic traits were investigated in detail. FSL #83, expressing the brassinosteroid biosynthetic pathway gene (CPD), displayed significant higher in internode length, stem count, flower number and above-ground dry matter weight compared to non-transgenic SR. On the other hand, FSL #121 expressed Asparaginyl-tRNA synthetase (SYNC1) gene exhibited increased plant height and the number of stem nodes. The information on candidate genes related on biomass obtained in this study can be expected to improve of other legume crops in the future.

All authors declare that they have no conflict of interest.