In this study, 27, 18, 17, 16, and 23 JRL protein members were identified from Oryza sativa (O. sativa), Brachypodium distachyon (B. distachyon), Zea mays (Z. mays), Sorghum bicolor (S. bicolor), and Setaria italica (S. italica), respectively ( Figure 1 ; Supplementary Table 1 ). We found that O. sativa has more JRL genes than S. italica, S. bicolor, B. distachyon and Z. mays, implying a gene expansion of the JRL gene family among different Gramineae species. Further, we named these JRL genes according to their location on the chromosomes: OsJRL1-OsJRL27; BdJRL1-BdJRL18; ZmJRL1-ZmJRL17; SbJRL1-SbJRL16 and SiJRL1-SiJRL23 ( Figure 1 ). The JRL genes of O. sativa are distributed on 9 chromosomes ( Figure 1A ). BdJRL genes ( Figure 1B ) and AtJRL genes ( Figure 1F ) are focusly distributed on 4 chromosomes. While ZmJRL genes and SiJRL genes are distributed on 7 chromosomes, respevtively ( Figures 1C, E ), SbJRL genes are distributed on chromosomes 1, 2, 3, 5, and 9 ( Figure 1D ). And these JRL genes are often distributed together in clusters ( Figure 1 ).

To investigate the evolutionary mechanism of JRL proteins in Gramineous, we used the Neighbor-Joining (N-J) method to construct an evolutionary tree including 46 AtJRL, 27 OsJRL, 23 SiJRL, 16 SbJRL, 18 BdJRL and 17 ZmJRL proteins. As shown in Figure 2 , these JRL proteins are divided into 8 subgroups. The JRL-IV group contains the most members, including 12 OsJRL, 1 AtJRL, 10 SiJRL, 12 ZmJRL, 8 SbJRL, and 2 BdJRL members. In contrast, JRL-I contains the fewest JRL members. Interestingly, the JRL-III and JRL-VI groups only include the Gramineous JRL proteins. And JRL-VIII is unique to Arabidopsis, containing 33 AtJRL proteins (71.7%). In addition, we found that the ZmJRL is lost in the group of JRL-III and JRL-V. Meanwhile, both JRL-IV and JRL-VII contain members of Gramineae JRL and Arabidopsis JRL, suggesting that JRL members in these two groups may function conserved between Arabidopsis and Gramineae ( Figure 2 ).

The diversity of gene structure can reflect the evolutionary processes of gene family. We analyzed the JRL gene structure, protein motif and conserved domain in O. sativa, B. distachyon, Z. mays, S. bicolor, S. italica and A. thaliana. The protein motif analysis showed that nearly all JRL proteins contain motif 3, motif 4, motif 8, motif 9 and motif 10, indicating that these motifs may be conserved for the functions roles of JRL proteins. We observed that JRL members of the same subgroup exhibit similar conserved motifs ( Figures 3A, B ). In contrast to the other six subgroups, members of the JRL-VII and JRL-VIII subgroups contain two or three Motif 1 and Motif 2 in tandem. While JRL-VIII comprises only Arabidopsis members, JRL-VII includes both Gramineae JRL genes and AtJRL genes. These suggests that the members of these two subgroups may possess unique functions in the evolutionary process, warranting further investigation.

We found that AtJRL28, AtJRL29, SiJRL5, and BdJRL7 contain two “Jacalin domain”, while some JRL proteins like SiJRL8, ZmJRL14, OsJRL12, and SbJRL15 possess three “Jacalin domain”. Interestingly, we found the members in JRL-VII and JRL-VIII subgroups contain 1-5 Jacalin domains. However, the conserved domain of S. bicolor is relatively simple, comprising only three types of domains: Jacalin, Dirigent and PKc_like superfamily. In contrast, the OsJRL members contain more complex conserved domains ( Figures 3A, C ). These suggests that there are both functional conservation and functional differentiation among the Gramineae JRL family members. Moreover, most AtJRL proteins that only contained Jacalin domain and no other domains ( Figure 3 ). Notably, AtJRL proteins are more likely to have multiple tandem Jacalin domain repeats. For example, AtJRL4 is composed of 5 tandem Jacalin domains ( Figures 3A, C ). It is noteworthy that a dirigent domain, which is the key domain of DIR proteins (Gong et al., 2023), was found at the N-terminal of the Gramineous JRL proteins, but was not present in Arabidopsis.

The number of exons in JRL genes ranged from 2 to 14 ( Figures 3A, C, D ). In O. sativa, the number of introns varied from 1 to 12. However, AtJRL genes exhibited a narrower intron range of 1-6 ( Figure 3D ). Furthermore, we observed that members of the same subgroup tend to exhibit similar gene structures. Previous studies have indicated that in the same gene family, genes with multiple introns play significant roles in stress resistance compared to those without introns (Shaul, 2017). The considerable variation in gene structure among Gramineous plants suggests potential substantial differences in gene function.

During evolution, tandem duplication (TD) and whole genome duplication (WGD) have played crucial roles in the expansion of gene families. In this study, we analyzed the duplication events of the JRL gene family members in O. sativa, Z. mays, S. italica, S. bicolor, B. distachyon and Arabidopsis. As shown in Figure 4 ; Supplementary Table 2 , we identified 61 TD and 18 WGD genes. Specifically, we found 6, 7, 6, 7, 8, and 27 TD genes, and 0, 4, 2, 4, 4 and 3 WGD genes in O. sativa, S. bicolor, B. distachyon, Z. mays, S. italica, and Arabidopsis, respectively. The TD genes of AtJRL6-13 ( Figure 4 ) are predominantly located in Group VIII of Figure 2 . Moreover, some TD genes of O. sativa, Z. mays, S. italica, S. bicolor and B. distachyon were grouped into the same subfamily ( Figure 2 ), suggesting that the expansion of JRL gene families in Z. mays, S. italica, S. bicolor and B. distachyon is primarily driven by the significant expansion of a specific subfamily. It can be inferred that both TD and WGD events jointly promote the expansion of JRL gene families in O. sativa, Z. mays, S. italica, S. bicolor, B. distachyon and Arabidopsis, with TD playing a a major dominant role.

Further, we calculated the ratio of Ka/Ks on TD and WGD gene pairs. And except for one gene pair in rice was larger than 1, the ratio of Ka/Ks of all other replicated gene pairs were smaller than 1 ( Figure 5A ). Additionally, we evaluated the appearance time of these TD and WGD genes. And the divergence time of TD genes in the ZmJRL, BdJRL, SiJRL, OsJRL, SbJRL and AtJRL ranged from 2.77 to 62.68, while WGD genes in these species ranged from 31.15 to 80.44 ( Figure 5B ). These results hint that most duplicate genes of the JRL family were under purification selection, and TD duplication events might contributed chiefly to their evolution in O. sativa, A. thaliana, S. italica, B.distachyon, S.bicolor and Z. may.

To further investigate the evolutionary mechanism of the JRL gene, we conducted a collinearity analysis across O. sativa, B. distachyon, S. bicolor, S. italica, Z. mays, and A. thaliana. As shown in Figure 6 , the number of collinear gene pairs between O. sativa and the other species are follows: 9 with B. distachyon, 8 with S.bicolor, 8 with S. italica, 6 with Z. may and 0 with A. thaliana. It is noteworthy that some OsJRL genes exhibit collinear relationships with at least two genes. For example, OsJRL4 is collinear with SbJRL6 and SbJRL16; OsJRL12 is collinear with SbJRL7, SbJRL15, ZmJRL10 and ZmJRL15.. These findings suggest that these genes may represent significant genetic resources. Notably, the genetic relationship between O. sativa and B. distachyon is closer than that observed among S. italica, Z. may, and S.bicolor. However, no co-linear gene pairs between O. sativa and A. thaliana was found, which demonstrates the function differences between monocotyledonous and dicotyledonous plants, to some extent.

To better understand the function, we analyzed the cis-elements of 147 JRL genes using their 2k-length promoter sequences. Our results revealed a strong connection between the JRL gene and various processes, such as meristem expression, circadian rhythm, root-specific and seed-specific regulatory elements. In addition, the JRL gene displayed a wide array of responsiveness to plant hormones, indicating its involvement in a complex hormone signaling network. And the most prevalent observed elements included abscisic acid response elements (ABRE) followed by jasmonic acid response elements (CGTCA-motif and TGACG-motif), implying the significant role of the JRL gene in coping with abiotic stress. The existence of light-responsive elements such as G-box and I-box implies that JRL genes might play a role in photosynthesis. The discovery of transcription factor binding sites, including W-box, indicates that multiple transcription factors may regulate the JRL genes’ transcription. Additionally, we observed that the cis-elements of MBSI associated with flavonoid biosynthesis was found in A. thaliana, O.sativa, and Z. mays, but not in B. distachyon, S. italica and S. bicolor. On the contrary, the cis-elements of motif I associated with root specific was found in S. italica and S. bicolor, but not in other four species ( Figure 7 ; Supplementary Table 3 ). Moreover, members of the same subgroup have similar cis-acting elements ( Figure 7 ). These results indicate that the JRL genes may simultaneously have functional consistency and differences.

To enhance our understanding about the potential function of the JRL genes, we analyzed the tissue expression patterns of OsJRL and ZmJRL genes. Our findings revealed that OsJRL2/8/9/17/21/22 and ZmJRL1/6/13/17 were not expressed in any of the four examined tissues ( Figure 8 ; Supplementary Table 4 ). And 44.44% of OsJRL genes and 17.65% of ZmJRL genes exhibited high expression levels in roots, while 11.11% of OsJRL genes and 29.41% of ZmJRL genes were highly expressed in flowers. OsJRL3/13/14/15/23/26 and ZmJRL5/12 were exclusively expressed in roots, whereas OsJRL7 and ZmJRL7 were exclusively expressed in flowers. Additionally, OsJRL4/5/12/16 and ZmJRL10/15 were highly expressed in stems; OsJRL10/24 and ZmJRL4/11 are highly expressed in leaf ( Figure 8 ). Interestingly, the collinear gene pairs OsJRL6/ZmJRL16 were both highly expressed in their inflorescence. And the collinear genes OsJRL12, ZmJRL10 and ZmJRL15 demonstrated high expression levels in stems ( Figure 8 ). However, the tissue expression patterns of the collinear gene pairs OsJRL20/ZmJRL3 exhibited differently: OsJRL20 was highly expressed in roots, while ZmJRL3 showed broad expression across root, flower, stem, and leaf.

For further investigation, we explored the expression levels of ZmJRLs and OsJRLs under abiotic stress by mining transcriptome data. As shown in Figure 9 ; Supplementary Table 5 , the expression levels of OsJRL1/4/27 genes were significantly up-regulated, while OsJRL6/9/17/18/20/24 genes were down-regulated under drought stress. Under salt, heat and cold stresses, OsJRL5/14 genes showed similar up-regulated expression patterns. Interestingly, some OsJRL genes displayed opposing expression patterns under different stress treatments. For instance, the expression levels of OsJRL15/18/20/23 were up-regulated under heat stress, but down-regulated under drought, salt and cold stresses ( Figure 9A ). In addition, we observed that the expression levels of ZmJRL6/9/11/15 genes were up-regulated under salt stress, with ZmJRL6 showing an extremely significant up-regulation to approximately 51-fold. The expression level of the ZmJRL3 gene was slightly up-regulated under cold stress, but it did not respond to drought, salt and heat stress ( Figure 9B ). Moreover, we note a descending trend in the expression levels of ZmJRL1 and ZmJRl10 under salt stress.

To further explore the potential function of the JRL gene, we randomly selected six OsJRL genes that were expressed in roots and responsive to abiotic stress. The mRNA levels of these six genes were assessed using RT-qPCR following treatment with 30% PEG6000 and 150 mM NaCl, respectively ( Figure 10 ). The transcription level of OsJRL4 was significantly up-regulated under PEG6000 treatment and peaking at 24 h, which was similar to its response under salt stress ( Figures 10A, G ). The expression levels of OsJRL12 and OsJRL26 were down-regulated under 30% PEG6000 treatment ( Figures 10B, F ), while OsJRL15 exhibited a significant up-regulation ( Figure 10D ). Under salt stress, the expression levels of OsJRL12, OsJRL14 and OsJRL26 were significantly up-regulated ( Figures 10H, I, L ), whereas OsJRL15 was inhibited ( Figure 10J ). Interestingly, the expression level of OsJRL26 was initially increased and then decreased under salt stress ( Figure 10L ).

The plant material utilized in this study was the rice variety Zhonghua 11 (ZH11). Rice seeds were initially soaked and incubated in purified water overnight in a dark incubator set at 28°C. After two days, uniform seedlings were selected and transferred to be grown in Kimura nutrient solutions, and they were grown under a photoperiod of a 10-hour light/14-hour darkness, with a relative humidity of 50%. Following a 10-day cultivation period, various stress treatments were administered. Drought stress was simulated by application of 30% polyethylene glycol (PEG6000), while salt stress was treated using 150 mM NaCl. Plant roots were collected from both the control and experimental groups at 0, 12, 24, and 48 hours post-treatment. The samples were rapidly frozen in liquid nitrogen, and stored at –80°C for future analysis. The experiment was conducted with three biological replicates.

The species selected for this study include Oryza sativa (O. sativa), Brachypodium distachyon (B. distachyon), Sorghum bicolor (S. bicolor), Zea mays (Z. mays), Setaria italica (S. italica) and Arabidopsis thaliana (A. thaliana). The genome files and gene structure annotation files of the above-mentioned species were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/) (Goodstein et al., 2012). The sequences of the JRL protein family in A. thaliana were downloaded from TAIR (https://www.Arabidopsis.org/).

The protein sequences of AtJRLs were used as reference sequences for bidirectional BLAST alignment (E-value < 1e^-5) to identify potential candidate JRL proteins across various organisms. The unique domain file (PF01419) specific to the JRL protein family was obtained from the Pfam website (http://pfam-legacy.xfam.org/) (Mistry et al., 2021). Subsequently, HMMER 3.0 software was employed to screen for potential candidate JRL family members within the five species under investigation (Potter et al., 2018). The presence of the conserved JRL domain in the candidate proteins was confirmed using NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART (http://smart.embl-heidelberg.de/). Proteins that lacked the JRL domain were excluded from the candidate list based on this analysis.

The JRL protein sequences that we have identified from Gramineae plants were used to construct an evolutionary tree by MEGA7.0 software and neighbor joining (NJ) method (Kumar et al., 2016), with parameter settings of poisson model, pair wise deletion, and 1000 bootstrap replicates.

The genome location of the JRL genes in each species was obtained from their gene structure annotation files (GFF3), named according to their order on chromosomes, and visualized by TBtools. Gene replication events were explored by the Multiple Collinearity Scan Toolkit (MCScan), with parameters set to default (Wang et al., 2012). Using rice as a reference, collinearity analysis was conducted with A. thaliana, B. distachyon, S. bicolor, Z. mays and S. italica. The results were visualized by TBtools software (Chen et al., 2020). The ratio of Ka/Ks for tandem replication and segmental replication gene pairs were calculated through TBtools software (Chen et al., 2020).

Gene expression data from different tissues of rice and maize under abiotic stress were obtained from the Plant Public RNA-seq Database (PPRD) (http://ipf.sustech.edu.cn/pub/plantrna/) (Yu et al., 2022). The gene expression was calculated using FPKM values. To highlight gene expression changes, we use Log₂ converted values with a multiple change of 1+FPKM (treatment group)/1+FPKM (control group). Heat maps were generated by TBtools software.

The primer 5.0 software was used to design specific primers for JRL genes in this study ( Supplementary Table 6 ). Total RNA was extracted using the KKFast Plant RNApure Kit (ZOMANBIO, ZP405K-2). The cDNA was synthesized by EX RT kit (gDNA remover) (ZOMANBIO, ZR108-3). The quantitative real-time PCR (RT-qPCR) system program was performed according to the previous research (Duan et al., 2023). The gene expression was analyzed by the 2^−ΔΔCT method as used in the previous study (Xue et al., 2024).