Genome-wide identification and expression analysis of late embryogenesis abundant (LEA) genes reveal their potential roles in somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana)

Ying Li; Shuaizheng Qi; Siyuan Chen; Hongxuan Li; Ting Zhang; Fen Bao; Dingju Zhan; Zhenwu Pang; Jinfeng Zhang; Jian Zhao; Ying Li; Shuaizheng Qi; Siyuan Chen; Hongxuan Li; Ting Zhang; Fen Bao; Dingju Zhan; Zhenwu Pang; Jinfeng Zhang; Jian Zhao

doi:10.48130/FR-2023-0012

Figures (8) Tables (4)

Figure 1.
Maximum likelihood phylogenetic tree of 79 LsfLEA genes and 51 AtLEA genes. The phylogenetic tree was constructed using MEGA 7 software with 1000 bootstrap replicates. We used the online site iTOL for evolutionary tree beautification, and the 9 major groups are marked with different colored backgrounds.
Figure 2.
(a) Motif analysis of LsfLEA genes from hybrid sweetgum. The conserved motifs of each group on the right side were identified by the MEME web server. Different motifs are represented by different colored boxes (b) Gene structure distribution map of LsfLEA.
Figure 3.
Chromosomal localization of the LsfLEA genes and gene duplication events. (a) Chromosomal localization of hybrid sweetgum LsfLEA gene. (b) Numbers of LsfLEA genes on each chromosome in hybrid sweetgum. (c) Duplicate pairs of the LsfLEA genes in hybrid sweetgum. The red lines represent collinear pairs of the LsfLEA genes.
Figure 4.
Cis-acting elements analysis of LsfLEA genes. The patterns in different colors on the right represent different cis-elements.
Figure 5.
The cultures during hybrid sweetgum somatic embryogenesis and different tissues. (a) NEC: non-embryogenic callus. (b) EC: friable-embryogenic callus. (c)-(f) Somatic embryos at different developmental stages globular embryo (GE), heart-shaped embryo (HE), torpedo-shaped embryo (TE), cotyledonal embryo (CE). (a)-(f) Bars = 1000 μm. (g) Different tissues in root (R), stem (S), and leaf (L) of hybrid sweetgum.
Figure 6.
Heatmap of the differentially expressed 79 LsfLEA genes during hybrid sweetgum SE and different tissues. The heatmap was clustered by TBtools software. The heatmap indicates the gene expression level by Log2[FPKM] with a color scale, each row represents a single gene, the IDs are indicated to the outside of fan, and each circle represents a sample. NEC: non-embryogenic callus; EC: friable-embryogenic callus; GE: globular embryo; HE: heart-shaped embryo; TE: torpedo-shaped embryo; CE: cotyledonal embryo; R: root; S: stem; L: leaf.
Figure 7.
Transcriptome expression (FPKM value) and qRT-PCR value of six LEA family genes in hybrid sweetgum at different stages during somatic embryogenesis. Relative expression values were calculated using the 2^−ΔΔCᴛ method with apple EF1-α as a housekeeping gene. NEC: non-embryogenic callus; EC: friable-embryogenic callus; GE: globular embryo; HE: heart-shaped embryo; TE: torpedo-shaped embryo; CE: cotyledonal embryo; R: root; S: stem; L: leaf.
Figure 8.
Subcellular localization of LsfLEA 1-3 protein in N. benthamiana leaves. (a) GFP empty vector in bright field. (b) Empty vector in GFP green fluorescence. (c) Empty vector in RFP red fluorescence. (d) Empty vector in merge. (e) LsfLEA 1-3 protein in bright field. (f) LsfLEA 1-3 protein in GFP green fluorescence. (g) LsfLEA 1-3 protein in RFP red fluorescence. (h) LsfLEA 1-3 protein in merge.

Medium	Composition
Induction/ proliferation	Basic medium^[37] + 1 mg 2, 4-D + 0.5 mg 6-BA + 40 g/L sucrose + 1 g/L enzymatic hydrolyzed casein 2.6−2.7 g/L vegetable gel, pH adjusted to 5.6~5.7
Mature	Basic medium + 40 g/L sucrose + 6 g/L vegetable gel +5% PEG, pH was adjusted to 5.6~5.7
Germination	Basic medium + 40 g/L sucrose + 2.6-2.7 g /L vegetable gel, pH was adjusted to 5.6~5.7

Table 1.

Medium used in the experiment.

Gene_ name	Gene_id	Primer sequence
LsfLEA1-1	EVM0027957	F: TATTGGGGTGATGACGGTCC R: TTCCTCTGCGTGGCCATATC
LsfLEA3-3	EVM0021267	F: ATACTCGGCGGCATCACAAG R: ACGTCAATCTCTTCCGCACG
LsfLEA6-2	EVM0005932	F: TGGAGGACTACAAGCGTCAAG R: CACCACCGGAAAGAGTGGG
LsfDHN-2	EVM0003818	F: CCAATTGGGTTGGAAACCGTC R: TCACCGAGCTAGAGCTTGAAC
LsfLEA1-3	EVM0000925	F: AGGAGAAGTTGAGCGACATGG R: TTTGCTTGAGCTTCCTTCGC
LsfSMP-1	EVM0023588	F: ATCGTGATCTGACCGGCATC R: CATCAACCACCACCACATGC
EF1-r-07041	/	F: ACTGCACGGTCATTGATGCT R: AAAGCATGCTCACGGGTCTG

Table 2.

Primer sequence information of qRT-PCR.

Name	Gene ID	Family	Pfam ID	Length	kD	pI	GRAVY	Subcellular localization
LsfLEA1-1	EVM0027957	LEA1	PF03760	191	19.36702	6.8	−0.865	Cytoplasm. Nucleus.
LsfLEA1-2	EVM0018353	LEA1	PF03760	120	13.7979	9.25	−0.863	Nucleus.
LsfLEA1-3	EVM0000925	LEA1	PF03760	169	18.18858	8.77	−0.731	Nucleus.
LsfLEA1-4	EVM0022193	LEA1	PF03760	164	16.51795	8.83	−0.946	Cytoplasm. Nucleus.
LsfLEA1-5	EVM0016558	LEA1	PF03760	107	12.32725	9.33	−0.769	Nucleus.
LsfLEA2-1	EVM0002770	LEA2	PF03168	239	26.42419	10	−0.094	Cell membrane. Cell wall.
LsfLEA2-2	EVM0027034	LEA2	PF03168	205	22.97259	9.84	−0.017	Chloroplast.
LsfLEA2-3	EVM0005964	LEA2	PF03168	177	19.27253	10.27	0.042	Chloroplast.
LsfLEA2-4	EVM0015809	LEA2	PF03168	190	21.8101	10.03	−0.482	Chloroplast.
LsfLEA2-5	EVM0019694	LEA2	PF03168	214	23.44119	9.91	−0.121	Cell membrane. Nucleus.
LsfLEA2-6	EVM0004956	LEA2	PF03168	210	23.12978	9.97	−0.186	Chloroplast.
LsfLEA2-7	EVM0027836	LEA2	PF03168	219	24.18402	9.73	−0.195	Chloroplast.
LsfLEA2-8	EVM0001803	LEA2	PF03168	237	27.18655	9.15	−0.103	Chloroplast.
LsfLEA2-9	EVM0020637	LEA2	PF03168	178	19.33671	10.25	0.086	Chloroplast.
LsfLEA2-10	EVM0015055	LEA2	PF03168	184	19.86985	9.92	−0.223	Chloroplast. Nucleus.
LsfLEA2-11	EVM0017871	LEA2	PF03168	212	23.53223	10	−0.157	Chloroplast. Nucleus.
LsfLEA2-12	EVM0018801	LEA2	PF03168	260	28.41312	9.01	0.133	Chloroplast.
LsfLEA2-13	EVM0013310	LEA2	PF03168	198	21.34478	9.37	0.407	Cell membrane.
LsfLEA2-14	EVM0010683	LEA2	PF03168	215	23.44427	9.88	−0.083	Cell membrane. Nucleus.
LsfLEA2-15	EVM0023593	LEA2	PF03168	201	22.72988	10.05	0.109	Chloroplast. Nucleus.
LsfLEA2-16	EVM0028234	LEA2	PF03168	188	20.34671	10.27	−0.001	Chloroplast.
LsfLEA2-17	EVM0003550	LEA2	PF03168	151	16.49626	5.58	0.077	Chloroplast. Nucleus
LsfLEA2-18	EVM0008005	LEA2	PF03168	265	29.16785	9.35	−0.066	Chloroplast.
LsfLEA2-19	EVM0007435	LEA2	PF03168	267	29.08778	8.38	−0.215	Cell membrane. Cell wall. Nucleus.
LsfLEA2-20	EVM0028118	LEA2	PF03168	223	25.25915	10.05	−0.122	Chloroplast. Mitochondrion.
LsfLEA2-21	EVM0001898	LEA2	PF03168	215	23.68493	10.14	−0.021	Nucleus.
LsfLEA2-22	EVM0018273	LEA2	PF03168	232	24.94073	9.3	0.113	Cell membrane. Chloroplast.
LsfLEA2-23	EVM0025800	LEA2	PF03168	189	20.97945	10.01	−0.074	Chloroplast. Nucleus. Peroxisome.
LsfLEA2-24	EVM0013583	LEA2	PF03168	264	29.43273	8.93	−0.048	Cell wall. Nucleus.
LsfLEA2-25	EVM0020355	LEA2	PF03168	306	34.40006	9.47	−0.383	Cell membrane. Chloroplast.
LsfLEA2-26	EVM0011840	LEA2	PF03168	214	23.38122	10.14	−0.056	Chloroplast.
LsfLEA2-27	EVM0023465	LEA2	PF03168	215	23.46828	9.91	−0.064	Cell membrane. Nucleus.
LsfLEA2-28	EVM0021483	LEA2	PF03168	262	29.00554	6.51	−0.288	Cell wall.
LsfLEA2-29	EVM0024390	LEA2	PF03168	213	23.36631	9.94	−0.035	Nucleus.
LsfLEA2-30	EVM0024154	LEA2	PF03168	215	23.32817	9.88	−0.031	Chloroplast.
LsfLEA2-31	EVM0007128	LEA2	PF03168	244	27.2663	10.2	−0.063	Chloroplast.
LsfLEA2-32	EVM0007725	LEA2	PF03168	259	28.26897	9.17	0.09	Chloroplast.
LsfLEA2-33	EVM0019270	LEA2	PF03168	223	24.39271	9.61	0.041	Cell wall. Chloroplast.
LsfLEA2-34	EVM0011791	LEA2	PF03168	210	23.90993	9.78	0.129	Cell membrane. Chloroplast.
LsfLEA2-35	EVM0015677	LEA2	PF03168	191	20.8606	9.28	0.33	Chloroplast.
LsfLEA2-36	EVM0011399	LEA2	PF03168	209	22.83086	9.98	0.197	Chloroplast.
LsfLEA2-37	EVM0020583	LEA2	PF03168	151	16.45712	5.04	0.054	Cell membrane. Nucleus.
LsfLEA2-38	EVM0011512	LEA2	PF03168	213	23.27806	9.97	−0.17	Chloroplast. Nucleus
LsfLEA2-39	EVM0000290	LEA2	PF03168	251	28.02687	9.56	−0.076	Chloroplast.
LsfLEA2-40	EVM0006920	LEA2	PF03168	269	29.83144	10.03	−0.246	Nucleus.
LsfLEA2-41	EVM0027367	LEA2	PF03168	253	27.51712	10.3	−0.089	Cell membrane. Cell wall.
LsfLEA2-42	EVM0005315	LEA2	PF03168	311	34.5181	9.07	−0.186	Cell wall. Nucleus.
LsfLEA2-43	EVM0025389	LEA2	PF03168	178	19.27267	10.25	0.108	Chloroplast.
LsfLEA2-44	EVM0000222	LEA2	PF03168	186	21.21822	7.87	−0.163	Cell membrane. Chloroplast.
LsfLEA2-45	EVM0018178	LEA2	PF03168	212	23.1178	9.91	−0.134	Cell membrane. Cell wall.
LsfLEA2-46	EVM0005757	LEA2	PF03168	178	19.26954	10.06	0.087	Chloroplast.
LsfLEA2-47	EVM0016553	LEA2	PF03168	209	22.80349	10.14	−0.098	Chloroplast.
LsfLEA2-48	EVM0016432	LEA2	PF03168	210	23.00723	9.83	0.226	Mitochondrion. Nucleus.
LsfLEA2-49	EVM0021824	LEA2	PF03168	308	34.60045	9.64	−0.337	Cell membrane. Chloroplast.
LsfLEA2-50	EVM0023803	LEA2	PF03168	214	23.29603	9.88	−0.102	Cell membrane. Cell wall.
LsfLEA2-51	EVM0017004	LEA2	PF03168	197	21.68226	10.22	−0.131	Chloroplast.
LsfLEA2-52	EVM0012352	LEA2	PF03168	217	24.03337	10.21	0.105	Chloroplast.
LsfLEA2-53	EVM0023248	LEA2	PF03168	225	24.9939	9.55	−0.009	Cell membrane. Chloroplast.
LsfLEA2-54	EVM0006284	LEA2	PF03168	213	23.40402	9.56	−0.175	Chloroplast. Cytoplasm. Nucleus.
LsfLEA2-55	EVM0008344	LEA2	PF03168	315	34.64837	4.75	−0.403	Nucleus.
LsfLEA2-56	EVM0022790	LEA2	PF03168	253	28.07601	10.17	−0.1	Cell membrane. Cell wall.
LsfLEA2-57	EVM0012837	LEA2	PF03168	191	21.15586	9.58	0.26	Chloroplast.
LsfLEA3-1	EVM0028438	LEA3	PF03242	101	10.79223	9.88	−0.222	Chloroplast. Nucleus.
LsfLEA3-2	EVM0019119	LEA3	PF03242	94	10.47005	9.37	−0.418	Chloroplast.
LsfLEA3-3	EVM0021267	LEA3	PF03242	121	13.48961	10.28	−0.668	Chloroplast.
LsfLEA4-1	EVM0019164	LEA4	PF02987	212	23.759	5.42	−1.533	Nucleus.
LsfLEA4-2	EVM0021387	LEA4	PF02987	323	36.9282	9.53	−1.53	Nucleus.
LsfLEA5-1	EVM0020631	LEA5	PF00477	94	9.93673	5.48	−1.26	Nucleus.
LsfLEA5-2	EVM0022920	LEA5	PF00477	114	12.14812	5.53	−1.278	Nucleus.
LsfLEA5-3	EVM0002005	LEA5	PF00477	114	12.07605	5.91	−1.251	Nucleus.
LsfLEA6-1	EVM0004641	LEA6	PF10714	89	9.40021	4.75	−0.939	Nucleus.
LsfLEA6-2	EVM0005932	LEA6	PF10714	89	9.37028	5.77	−1	Nucleus.
LsfSMP-1	EVM0023588	SMP	PF04927	259	26.66671	4.66	−0.253	Nucleus.
LsfDHN-1	EVM0028566	Dehydrin	PF00257	290	32.91761	5.23	−1.388	Nucleus.
LsfDHN-2	EVM0003818	Dehydrin	PF00257	174	18.52736	8.01	−1.126	Cytoplasm.
LsfDHN-3	EVM0011412	Dehydrin	PF00257	174	18.62131	9.45	−1.31	Cytoplasm.
LsfDHN-4	EVM0018312	Dehydrin	PF00257	193	21.77947	5.56	−1.388	Nucleus.
LsfDHN-5	EVM0008852	Dehydrin	PF00257	1891	21.36301	5.46	−1.348	Nucleus.
LsfDHN-6	EVM0003289	Dehydrin	PF00257	155	16.54385	9.1	−1.369	Cytoplasm.

Table 3.

The characteristics of LsfLEA genes.

Motif number	Motif sequences
1	NTRFVAQVTVKNTNFGPYKFDNTTATFTYGGMTVGZVVIPKGKAGARSTK
2	SDISSGILKLSSQAKLSGKVELMFVMKKKKSAEMNCTLTFBLSAKALQAL
3	RRIKIAAYIVAFVVFQSIIIAIFAVTVMKYRTPKFRLGKVTVETLTSTPA
4	VVALJLWLVFRPKRPKFTVNSVSVSSFNL
5	DEESATLQSKEFRRK
6	RNPNKKJSIYYDSIEASVYY
7	KIBVTVDLNSDALTS
8	QQILSSGSLPPFYQGRKNTTVLSTTLAG
9	FBVKVDARVRFKVGRWKTKRVGVRVSCDG
10	MAEKNQQVYPAAP
11	SAAEKKKGMKEKIKEKLPGYKAKAEEEKE
12	HEGRKAKEAQAKMEMHEAKARHAEEKLNAKQSHLYGHQVHE
13	PKRERRRGCCCCGCL
14	KGGQTRKEQLGTEGYQEMGRKGGLSTTDQSGGERAAELGVDIDESKF
15	HHPEEKKGILEKIKEKLPGQH
16	GNREQREELEARARQGETVVPGGTGGGSLEAQEHLAEGRS
17	PHHYHCSPIHHSRESSTSRFSASLKNAHHHKHSAPWKKMHRVVDVDDDD
18	RDEYGNPIQLTDERGNPVQLTDEYGNPMHLSGVATTHGTTA
19	LNLTFVMRSRAYILGKLVKSKFYKRIRCSVTLRGNQLGKLQNLTDSCIYQ
20	NDITMLDVGLKVPHNVLVTLVKDIGADWDIDYELEVGLTIDLPIIGNFTI
21	HIIGTMNQWWVSMDTMGRSLLELRPLYLY
22	WVPDPVTGYYRPENRAGEIDVAELRD
23	MHAKSDSEVTSLDASSPPRSPRRPLYYVQSPSQHDVEKMSY
24	NFMVEKVANIKKPEASVEDVDLKDVSRECIDYNAKVAVKNPYGHALPICE
25	MADQHFQCHEKEPRDRGMFDF
26	GTMVGGPRECKVRL
27	MADSAIRKTEDSPPSSKPSPNPTSKPVRHVVFSEIPCRPHK
28	IQQSQVDIGQEPKCSVKMFSFRLHTFLFI
29	EGGEEKKKKKGLKEKIKEKMACQGEEEVTEIPVDKCDNIVDAET
30	GMDKTKATMQZKVEKMTARDPIZKEMATZKKEAKKTEAELNKQEAREHNA
31	CCAWGCMIGF
32	EPQEKKEVEKPTLVEELRRSGSSSSSSSD
33	GLPLESSPYVKNSDLEDYKRKGYGTEGHLDPKPGRGAGGTDAP
34	SSQITGNWNVSFYV
35	CKVDLRIKIWKWTF
36	GHPTGGHQMSAMPGEGTWQPTW
37	YQVPLYGGVSVLGGARNHYEN
38	MEDQKKPVIGYPVQFYHP
39	NTAAAHVVQPPPVVHHQQQ
40	GKVGEYKDYAAEKAKETKDSALEKAREYKD

Table 4.

The motif sequences of LsfLEA genes.