Identification of LsGRAS genes in lettuce
A total of 59 LsGRAS genes were identified by homology search against the Arabidopsis GRAS sequences (Table 1). Meanwhile, all 59 genes were mapped to 9 different lettuce chromosomes (Fig. 1). The 59 LsGRAS1 genes were renamed from LsGRAS1 to LsGRAS59 according to chromosome names and locations. Basic characteristics of LsGRAS family members include the open reading frame (ORF) length, isoelectric point (pI), the protein molecular weight (MW), and the predicted subcellular localization. LsGRAS18 was the smallest protein with a length of 420aa, while LsGRAS34 was the largest protein with a length of 802aa. The MW of the proteins ranged from 47,516.82 to 87,207.15 Da, and the pI ranged from 4.9 (LsGRAS1 and LsGRAS6) to 8.6 (LsGRAS2). The predicted subcellular localization results showed 100% nuclear localization (Table 1).
The 59 LsGRAS genes were unevenly distributed on 9 chromosomes, among which the number of LsGRAS genes on chromosome 5 was the largest, with 11, and the number of LsGRAS genes on chromosome 4 was the least, with 4 (Fig. 1). It’s worth noting that 17 LsGRAS genes are at least one LsGRAS that is physically adjacent to another, such as LsGRAS2–LsGRAS3, LsGRAS14–LsGRAS15, LsGRAS31–LsGRAS32, LsGRAS34–LsGRAS35, LsGRAS39–LsGRAS40, LsGRAS41–LsGRAS43, and LsGRAS55–LsGRAS58. Those LsGRAS make up about 50% of the total genes.
Table 1
Detail information of LsGRAS
mRNA Namea | Gene Nameb | Gene IDc | Lengthd | PIe | MWf | SLg |
XM_023899939.2 | LsGRAS1 | LOC111904158 | 535 | 4.9 | 60331.9 | Nucleus. |
XM_023912371.1 | LsGRAS2 | LOC111916701 | 463 | 8.6 | 53145 | Nucleus. |
XM_023912370.2 | LsGRAS3 | LOC111916700 | 464 | 6.31 | 52883.18 | Nucleus. |
XM_023917371.2 | LsGRAS4 | LOC111921793 | 467 | 5.67 | 52701.63 | Nucleus. |
XM_023872476.2 | LsGRAS5 | LOC111875951 | 447 | 6.05 | 50251.7 | Nucleus. |
XM_023900832.1 | LsGRAS6 | LOC111905160 | 468 | 4.9 | 54276.87 | Nucleus. |
XM_023879739.2 | LsGRAS7 | LOC111883408 | 582 | 5.02 | 63282.65 | Nucleus. |
XM_023879795.2 | LsGRAS8 | LOC111883461 | 523 | 5.41 | 58477.35 | Nucleus. |
XM_023896081.2 | LsGRAS9 | LOC111900213 | 532 | 5.64 | 60046.82 | Nucleus. |
XM_023901493.2 | LsGRAS10 | LOC111905757 | 723 | 5.95 | 81370.2 | Nucleus. |
XM_023884628.2 | LsGRAS11 | LOC111888457 | 614 | 6.51 | 67805.15 | Nucleus. |
XM_023888978.2 | LsGRAS12 | LOC111892896 | 574 | 6.08 | 63627.1 | Nucleus. |
XM_023916486.2 | LsGRAS13 | LOC111920919 | 571 | 5.27 | 62495.69 | Nucleus. |
XM_023882048.2 | LsGRAS14 | LOC111885810 | 582 | 4.94 | 64787.71 | Nucleus. |
XM_023914966.2 | LsGRAS15 | LOC111919376 | 521 | 5.44 | 57716.52 | Nucleus. |
XM_023898897.2 | LsGRAS16 | LOC111903110 | 547 | 5.91 | 61708.32 | Nucleus. |
XM_023876680.2 | LsGRAS17 | LOC111880266 | 454 | 5.7 | 50433.69 | Nucleus. |
XM_023894263.2 | LsGRAS18 | LOC111898333 | 420 | 5.16 | 47516.82 | Nucleus. |
XM_042900756.1 | LsGRAS19 | LOC111884683 | 762 | 5.93 | 85207.15 | Nucleus. |
XM_023876376.2 | LsGRAS20 | LOC111879947 | 453 | 5.52 | 49702.44 | Nucleus. |
XM_023886497.2 | LsGRAS21 | LOC111890374 | 591 | 5.12 | 63529.61 | Nucleus. |
XM_023882398.2 | LsGRAS22 | LOC111886167 | 550 | 5.81 | 59325.03 | Nucleus. |
XM_023917098.2 | LsGRAS23 | LOC111921515 | 543 | 7.6 | 61064.99 | Nucleus. |
XM_023897710.2 | LsGRAS24 | LOC111901852 | 500 | 5.12 | 56090.62 | Nucleus. |
XM_023882047.1 | LsGRAS25 | LOC111885807 | 426 | 6.95 | 48433.9 | Nucleus. |
XM_023917341.2 | LsGRAS26 | LOC111921756 | 630 | 5.33 | 70435.78 | Nucleus. |
XM_023884834.2 | LsGRAS27 | LOC111888709 | 491 | 8.34 | 56173.88 | Nucleus. |
XM_023910585.2 | LsGRAS28 | LOC111914874 | 507 | 5.49 | 56480.36 | Nucleus. |
XM_023876955.2 | LsGRAS29 | LOC111880523 | 547 | 6.72 | 61357.13 | Nucleus. |
XM_023893864.2 | LsGRAS30 | LOC111897910 | 531 | 5.16 | 58419.36 | Nucleus. |
XM_023874860.2 | LsGRAS31 | LOC111878348 | 545 | 5.26 | 61012.01 | Nucleus. |
XM_023874675.2 | LsGRAS32 | LOC111878163 | 557 | 5.05 | 62755.68 | Nucleus. |
XM_023914310.2 | LsGRAS33 | LOC111918681 | 474 | 6.25 | 53731.38 | Nucleus. |
XM_023887787.2 | LsGRAS34 | LOC111891717 | 802 | 6.15 | 55501.91 | Nucleus. |
XM_023886757.2 | LsGRAS35 | LOC111890649 | 747 | 6.04 | 84426.2 | Nucleus. |
XM_023903060.2 | LsGRAS36 | LOC111907281 | 740 | 5.2 | 83659.51 | Nucleus. |
XM_023887613.2 | LsGRAS37 | LOC111891561 | 499 | 6.15 | 55501.91 | Nucleus. |
XM_023883122.2 | LsGRAS38 | LOC111886881 | 680 | 6.07 | 74775.27 | Nucleus. |
XM_023896302.2 | LsGRAS39 | LOC111900413 | 616 | 5.64 | 60046.82 | Nucleus. |
XM_023896303.2 | LsGRAS40 | LOC111900414 | 616 | 5.88 | 68694.99 | Nucleus. |
XM_023886550.2 | LsGRAS41 | LOC111890431 | 542 | 5.65 | 60825.06 | Nucleus. |
XM_023886553.1 | LsGRAS42 | LOC111890433 | 525 | 5.07 | 59047.92 | Nucleus. |
XM_042896049.1 | LsGRAS43 | LOC111895192 | 537 | 5.63 | 60602.17 | Nucleus. |
XM_023876909.2 | LsGRAS44 | LOC111880474 | 436 | 6.57 | 49181.56 | Nucleus. |
XM_023877780.2 | LsGRAS45 | LOC111881387 | 672 | 5.89 | 74919.83 | Nucleus. |
XM_023894356.2 | LsGRAS46 | LOC111898450 | 440 | 5.59 | 49587.6 | Nucleus. |
XM_023898665.2 | LsGRAS47 | LOC111902851 | 489 | 6.94 | 55144.69 | Nucleus. |
XM_023881251.2 | LsGRAS48 | LOC111884953 | 528 | 5.01 | 59119.49 | Nucleus. |
XM_023892671.2 | LsGRAS49 | LOC111896695 | 492 | 5.09 | 54974.1 | Nucleus. |
XM_023904280.2 | LsGRAS50 | LOC111908450 | 636 | 6.52 | 68912.59 | Nucleus. |
XM_023889368.2 | LsGRAS51 | LOC111893308 | 657 | 6.38 | 74389.54 | Nucleus. |
XM_023877838.2 | LsGRAS52 | LOC111881444 | 570 | 4.98 | 62044.82 | Nucleus. |
XM_023876767.2 | LsGRAS53 | LOC111880342 | 721 | 5.38 | 81983.71 | Nucleus. |
XM_023912753.2 | LsGRAS54 | LOC111917088 | 743 | 7.59 | 84084.93 | Nucleus. |
XM_023898073.2 | LsGRAS55 | LOC111902221 | 733 | 5.15 | 83122.89 | Nucleus. |
XM_023898074.2 | LsGRAS56 | LOC111902222 | 614 | 7.79 | 69694.81 | Nucleus. |
XM_023898086.2 | LsGRAS57 | LOC111902234 | 505 | 7.12 | 58006.6 | Nucleus. |
XM_023898076.2 | LsGRAS58 | LOC111902223 | 575 | 7.19 | 66079.86 | Nucleus. |
XM_023893945.2 | LsGRAS59 | LOC111897993 | 462 | 6.48 | 53238.42 | Nucleus. |
amRNA Name is miRNA expression sequence |
bGene named for their position on the chromosomes.
cAccession number of leaf lettuce locus ID.
dProtein length in amino acid.
eIsoelectric points.
fMolecular weight in Dalton.
gSubcellular localization.
Phylogenetic Analyses And Classifications Of Lsgras
To classify the phylogenetic relationships of lettuce LsGRAS proteins, we constructed a phylogenetic tree based on the identified LsGRAS in this study and Arabidopsis GRAS proteins from TAIR (https://www.arabidopsis.org/index.jsp) (Fig. 2). There is no specific identification sequence and motif classification for LsGRAS. Therefore, according to the topology structure of the phylogenic tree, LsGRAS can be divided into 9 main subfamilies, namely DELLA, HAM, LAS, LISCL, PAT1, SCL3, SCL4/7, SCR, and SHR. In contrast, LsGRAS55 was relatively independent and did not belong to any GRAS gene subfamilies. The PAT1 and SCL3 subfamily contained 9 members, which was the largest LsGRAS gene subfamily in this study, and the LISCL subfamily has one less gene member than the PAT1 subfamily. Coincidentally, the HAM and LISCL subfamilies each had 8 members. Among them, the DELLA subfamily herein contained 7 members. In addition, there were 7, 4, 3, and 4 LsGRAS gene members in the SHR, SCR, SCL4/7, and LAS subfamilies, respectively. Importantly, physically adjacent LsGRAS cluster in the same subfamily, suggests that the main evolutionary driver for LsGRAS expansion is through tandem duplication.
To Conserved Motifs And Gene Structure Of Lsgras
To further demonstrate the structures of the LsGRAS proteins, a schematic was built based on the LsGRAS-motif scanning result. Conserved protein motifs are associated with gene function and protein subcellular localization. We isolated 10 predicted conserved motifs (Fig. 3A) using LsGRAS and studied their distributions within LsGRAS. Phylogenetic analysis grouped LsGRAS into 9 subfamilies, which is consistent with the interspecific phylogenetic tree in Fig. 2. We found that 59 LsGRAS from subfamily DELLA included all 10 motifs and those motifs shared the same order. To further examine the evolutionary lineages of LsGRAS, we compared the gene structure. The results showed that phylogenetically close LsGRAS shared the same exon number, length, and composition. At the same time, By screening the corresponding genomic DNA sequences and the annotation files, the exon-intron patterns of the identified LsGRAS genes were obtained (Fig. 3B). The LsGRAS genes displayed one to seven exons (51 with one exon, 7 with two exons, and one with three exons) and lacked introns.
Evolutionary analyses of LsGRAS gene members
To explore the evolutionary clues for the lettuce LsGRAS gene family, we constructed 2 comparative syntenic graphs to display the lettuce of LsGRAS gene members between 2 representative species (Fig. 4). The 2 representative species contained Arabidopsis thaliana and Solanum Lycopersicum. Moreover, we analyzed the syntenic relationships between 59 lettuce LsGRAS gene members and those of Arabidopsis thaliana and Solanum Lycopersicum (Table S1), and found that there were 22 and 30 isogenies genes, respectively. Overall, the LsGRAS gene of the Solanum Lycopersicum gene consists of more pairs of isogenies than the Arabidopsis thaliana gene.
Transcriptome Analysis Under Exogenous Melatonin
To investigate the expression profiles of 59 LsGRAS members in lettuce under exogenous melatonin, we downloaded publicly available leaf transcriptome data from NCBI (Bio project PRJNA810911) [9] and conducted RNA-Seq analysis. The results showed that the changing pattern of the LsGRAS13 gene was consistent. In different periods, the expression level of high-temperature melatonin treatment was higher than that of non-exogenous melatonin treatment, with members of the DELLA subfamilies likely playing important roles (Fig. 5). The expression level of the LsGRAS52 gene was the highest, and the expression level of high-temperature melatonin treatment was higher than that of non-exogenous melatonin treatment at 6, 9, 15, and 18 days, while the opposite was true at 27 days. Five LsGRAS genes were not expressed in lettuce. In addition, 15 LsGRAS genes were insensitive to high-temperature stress.
To investigate the biological function of LsGRAS genes, according to the results of RNA-Seq, the LsGRAS13 gene with high transcription level and significant change regularity was selected for cis-element analysis. The 2 kb region upstream of the promoter start codon of the gene was isolated. Important cis-elements were identified (Table 2), including P-box, TATC-box (GA-responsive element), G-box, CTt-Motif, MRE (light-responsive element), Box 4 (auxin-responsive element), CGTCA-motif (MeJA-responsive element), MSA-like (cell cycle regulation) and TCA (salicylic-responsive element) components. This suggests that this gene regulates plant growth and development in response to light and hormones under high-temperature stress.
Table 2
Elements analysis of LsGRAS13
Site Name | Matrix sequence | Position | Strand | Motif annotation |
G-box | CACGAC | 796 | + | cis-acting regulatory element involved in light responsiveness |
G-box | CACGAC | 1052 | + | cis-acting regulatory element involved in light responsiveness |
circadian | CAAAGATATC | 1078 | - | cis-acting regulatory element involved in circadian control |
P-box | CCTTTTG | 1352 | + | gibberellin-responsive element |
TCA-element | CCATCTTTTT | 1320 | + | cis-acting element involved in salicylic acid responsiveness |
TGA-box | TGACGTAA | 1669 | - | part of an auxin-responsive element |
Box 4 | ATTAAT | 804 | + | part of a conserved DNA module involved in light responsiveness |
TCT-motif | TCTTAC | 1519 | - | part of a light responsive element |
MSA-like | (T/C)C(T/C)AACGG(T/C)(T/C)A | 1192 | + | cis-acting element involved in cell cycle regulation |
CGTCA-motif | CGTCA | 113 | + | cis-acting regulatory element involved in the MeJA-responsiveness |
CGTCA-motif | CGTCA | 1672 | + | cis-acting regulatory element involved in the MeJA-responsiveness |
CGTCA-motif | CGTCA | 1964 | + | cis-acting regulatory element involved in the MeJA-responsiveness |
TATC-box | TATCCCA | 1529 | + | cis-acting element involved in gibberellin-responsiveness |
GT1-motif | GGTTAAT | 1976 | + | light responsive element |
MRE | AACCTAA | 314 | - | MYB binding site involved in light responsiveness |