Comprehensive Sequence and Expression Profile Analysis of the Phosphate Transporter Gene Family in Soybean

doi:10.21203/rs.3.rs-1962440/v1

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Comprehensive Sequence and Expression Profile Analysis of the Phosphate Transporter Gene Family in Soybean

https://doi.org/10.21203/rs.3.rs-1962440/v1

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The family of phosphate transporters (PHTs) mediates the uptake and translocation of Pi inside the plants. However, little is known about transporters in soybean. Therefore, Searched the Genome Database for Soybean, 57 GmPHTs family members were identified in soybean, Phylogenetic analysis suggested that members of the PHTs gene family can be divided into six clades. Collinearity analysis revealed that most of the GmPHT genes shared syntenic relationships with PHTs members in Arabidopsis thaliana and that large segment duplication played a major driving force for GmPHTs evolution in addition to tandem duplication. Further analysis of the promoter revealed that light-responsive elements and abiotic stress-responsive elements were widely distributed within the promoter regions of GmPHT genes. Based on RNA-seq data, GmPHTs showed different expression patterns in roots and leaves of soybean treated with long-term low phosphorus and short-term low phosphorus, in addition, the expression levels of GmPHT genes can be regulated by drought stresses, it was implied that the induced expression of GmPHTs could promote phosphorus uptake and transport in soybean and thus adapt to low phosphorus and drought stress, which is the first step dissection of Pi transport system and probably refers to new roles of PHTs genes in soybean.

soybean

phosphate transporters

gene family

phosphorus stress

drought

Phosphorus (Pi) is a key nutrient for plant growth and development, is the second essential macronutrient required for plant growth and development alongside nitrogen ¹ and is also an essential component of fertilizers used to sustain modern agriculture because it helps quality and yield. Pi is part of important biomolecules and, in the form of phosphate, pyrophosphate, adenosine triphosphate, adenosine diphosphate, or adenosine monophosphate, involved in various metabolic functions and many living cell regulatory processes are P-dependent ². Globally, many soils are deficient in phosphate (Pi) ³, Pi concentrations within plant cells are typically 1000-times those outside ⁴. Therefore, plants must have specialized transport vehicles to transfer Pi from the soil to the cells to counter the large concentration gradient at the root-soil interface. The active phosphorus in the soil and the phosphorus used in various metabolic processes ultimately require direct uptake and transport through the "phosphate transporter protein (PHT)", a highly efficient plant root uptake and transport system, which is responsible for the transport of inorganic phosphorus in plants under the regulation of transcription factors. It has a different molecular structure, localization and function⁵. Genome sequence analyses and experimental evidence have indicated that plants contain numerous Pi transporter families, including PHT1, PHT2, PHT3, PHT4, and PHT5, which are distinguished by their protein sequences, structures, locations, and functions ^6-11.

The PHT1 family has been most extensively studied in plants, and the main pathway for Pi to enter plants from the soil is through the PHT1 family located on the plasma membrane, PHT type 1 protein is also defined as a major contributor to the Pi uptake system ¹². and each plant has several PHT1 transporter members that play a key role in P acquisition, transport and remobilization. The Arabidopsis AtPHT1 gene family contains nine members, The rice OsPHT1 gene family has 13 members ¹³. The first high H+/PI phosphate transporter identified in higher plants was AtPT1 from Arabidopsis ¹⁴. This gene has an important role in the uptake of phosphorus from the soil ¹⁵. Analysis of expressed sequence tags (ESTs) and genomic sequences revealed that nine genes in Arabidopsis are similar to AtPT1, with PHT1;1 transcript being the most abundant ¹⁶. Its overexpression increases Pi uptake in Arabidopsis ¹⁷. The PHT2 gene family contains one member each in Arabidopsis thaliana and rice¹³, and direct homologs have been characterized in wheat, alfalfa rhizobia and several Solanum species ¹⁴. Arabidopsis PHT2;1 is the first member of the PHT2 family to be identified, with OsPHT2;1 in rice being the putative low-affinity phosphate transporter gene involved in the accumulation and transport of Pi in the plant ¹⁸. The third family of plant Pi transporters is localized to the mitochondria, Three PHT3 genes have been identified in Arabidopsis ¹⁹ and six members identified in rice ¹³, and homologs have been cloned and partially characterized soybean, maize, and birch ^20,21. The Arabidopsis genome contains six PHT4 genes (PHT4;1 to PHT4;6), and chloroplast localization of PHT4;1, PHT4;2, PHT4;4, and PHT4;5 has been analyzed using GFP fusions or immunoblotting^22-24. In Arabidopsis, phosphate transporter proteins containing the SPX-MFS structural domain have been designated as members of the phosphate transporter protein family PHT5 or VPT. The results also confirmed that PHT5 could increase plant tolerance under low phosphorus stress. The results also confirmed that PHT5 could increase plant tolerance under low phosphorus stress ²⁵. GmPHT5 in soybean may be responsible for transporting phosphorus from root vascular tissues to nodules, especially under phosphorus deficiency stress, and therefore GmPT5 may be a key transporter in regulating nodule phosphorus homeostasis ²⁶.

Soybean (Glycine max (L.) Merr.) is an economical and agronomical crop, which has been adopted as a diet staple throughout many parts of the world ⁶, However low soil available phosphorus (P) is the leading nutritional factor limiting seed yield in soybean ^27,28. Previous studies that have reported that phosphorus addition can increase drought tolerance in soybean ^29,30. Phosphorus application increased nitrogen fixation rates in soybean ^31,32, which has been found to be sensitive to drought stress ^33-35. In production areas within China, drought is the most challenging stress for soybean ³⁶ and the non-renewable resource of P is predicted to have limited availability, soybean seed yield will be threatened by both water and P deficits in the future ³⁷. Therefore, it is urgent that researchers develop plants with enhanced efficiency of soil phosphorus use under such conditions, improve the capacity of soybean to absorb phosphorus. Because Pi is moved from the soil into plant cells in response to excess phosphate, genomic analyses have been conducted with Pi transporter families in Arabidopsis, rice and apple.

Currently, the study of phosphorus transport protein genes in Arabidopsis, rice and other plants has gained great progress, while PHTs have been rarely studied in soybean. Therefore, based on the published Arabidopsis thaliana genome, this study identified and characterized putative soybean PHTs family genes through phylogenetic analysis, collinearity analysis, motif, promoter element analysis, and investigated the expression profile of PHTs genes in response to low phosphorus and drought. Transcriptome sequencing was used to analyze the induction of expression of root and leaf phosphorus transporter-related genes in soybean by low phosphorus treatment. The objective was to present a foundation for further functional dissection of GmPHTs so that genetic engineering approaches can been applied in efforts to improve the efficiency of phosphate uptake by stressed soybean plants.

Identification of the PHTs family genes in soybean. Based on the homology with PHTs family members in Arabidopsis, a total of 57 PHTs genes were identified in the whole genome of soybean (Table 1). Each subfamily member also has its own characteristics. The differences in the isoelectric point and hydrophilicity of the subfamily proteins are relatively obvious, Except for the GmPHT5 subfamily, the isoelectric points of all subfamily members are basic, the isoelectric point of the GmPHT5 subfamily is acidic, at around 6. The GmPHT1, GmPHT2, GmPHT3, GmPHT4, and GmPHT5 subfamilies had positive hydrophilicity values ranging from 0.101 to 0.659, while the GmPHO1 subfamily had negative hydrophilicity values ranging from -0.255 to 0.064. In contrast, the hydrophilicity among the members of the GmPHT4 subfamily varied considerably, with GRAVY values ranging from 0.2-0.6. The differences in the basic physicochemical properties of the proteins reflect the differences in their physiological functions. In particular, the differences in isoelectric point and hydrophilicity exhibited among the members within the GmPHT4 subfamily herald the diverse functional differentiation among the members of the GmPHT4 subfamily.

Phylogenetic analysis of the GmPHTs genes. To determine the evolutionary relationships of the members of the GmPHTs gene family genes with those of the soybean species, we constructed a phylogenetic tree comprising 92 PHTs proteins, from soybean (57), Arabidopsis (35), based on a multialignment via MEGA 6. Our results showed that all the PHTs homologs could be classified into six clades. GmPHT1 subfamily (14 members), GmPHT2 subfamily (2 members), GmPHT3 subfamily (9 members), GmPHT4 subfamily (12 members), GmPHT5 subfamily (6 members), and GmPHO1 subfamily (14 members), respectively; which are highly similar to each other. In addition, almost all of the soybean PHTs genes appeared as pairs with the PHTs members in Arabidopsis in terms of phylogenetic relationships (Fig. 1).

Table 1 Localization and physicochemical properties of members of the GmPHTs protein gene family

Name	Gene Locus ¹	Gene Identifier ²	Chr	Locations	Length(bp)	Protein ID ¹	Length(aa)	pI	Mw(kDa)	GRAVY	TMHs	Predicted Subcellular Location
Name	Gene Locus ¹	Gene Identifier ²	Chr	Locations	Length(bp)	Protein ID ¹	Length(aa)	pI	Mw(kDa)	GRAVY	TMHs	WoLF PSORT		Softberry
GmPHT1.1	LOC100780201	Glyma.02G005800	2	634487..639193	1602	NP_001241164.1	533	8.31	58.5	0.309	12	vacu:7	Plasma membrane
GmPHT1.2	LOC100819445	Glyma.03G162800	3	37772793..37775073	1620	NP_001304639.2	539	8.67	59.3	0.291	11	plas:10	Plasma membrane
GmPHT1.3	LOC100797683	Glyma.07G222700	7	39866989..39873025	1536	NP_001241574.1	511	8.51	57.6	0.295	12	plas:11	Plasma membrane
GmPHT1.4	LOC100795623	Glyma.10G006700	10	662561..665361	1602	NP_001341396.1	533	8.54	58.4	0.324	12	plas:7	Plasma membrane
GmPHT1.5	LOC100786638	Glyma.10G036800	10	3233051..3236286	1566	NP_001304588.2	521	8.63	57.3	0.363	11	plas:7	Plasma membrane
GmPHT1.6	LOC100802365	Glyma.10G186400	10	41935063..41939261	1584	NP_001239971.1	527	8.91	58.1	0.397	11	plas:9	Plasma membrane
GmPHT1.7	LOC100802890	Glyma.10G186500	10	41946209..41950274	1611	NP_001240032.1	536	8.34	58.7	0.334	11	plas:10	Plasma membrane
GmPHT1.8	LOC100802261	Glyma.13G040200	13	12579481..12581070	1590	NP_001241400.1	529	8.82	58.7	0.316	10	plas:12	Plasma membrane
GmPHT1.9	LOC100805284	Glyma.14G123500	14	18527015..18528592	1578	NP_001241127.1	525	8.33	58.2	0.330	11	plas:11	Plasma membrane
GmPHT1.10	LOC100820250	Glyma.14G188000	14	45277372..45278961	1590	NP_001239765.1	557	8.18	61.2	0.232	12	plas:12	Plasma membrane
GmPHT1.11	LOC100803626	Glyma.19G164300	19	42521537..42524774	1620	NP_001254802.1	539	8.52	59.3	0.291	11	plas:10	Plasma membrane
GmPHT1.12	LOC100803834	Glyma.20G021600	20	2196534..2205616	1521	NP_001345390.1	506	8.63	56.8	0.333	11	plas:10	Plasma membrane
GmPHT1.13	LOC100792177	Glyma.20G204000	20	44094584..44098582	1611	NP_001240239.1	536	7.63	58.6	0.341	12	plas:11	Plasma membrane
GmPHT1.14	LOC100792711	Glyma.20G204100	20	44103550..44107427	1584	NP_001240957.1	527	8.93	58.1	0.386	11	plas:9	Plasma membrane
GmPHT2.1	LOC100787472	Glyma.08G282100	8	38707723..38713794	1728	XP_003530627.1	575	9.28	60.7	0.519	12	plas:7	Plasma membrane
GmPHT2.2	LOC100787349	Glyma.18G144100	18	23306379..23312679	1731	XP_003551229.1	576	9.31	60.5	0.502	12	plas:7	Plasma membrane
GmPHT3.1	LOC100788561	Glyma.01G157100	1	49479697..49484208	921	XP_003517120.1	306	9.13	33.8	0.167	1	chlo:5	Mitochondrial
GmPHT3.2	—	Glyma.02G065800	2	5845126..5847769	873	—	347	8.69	37.8	0.105	0	vacu:5	Mitochondrial
GmPHT3.3	LOC100791467	Glyma.05G201400	5	38507610..38510859	1080	XP_003524479.2	359	9.50	38.3	0.135	0	chlo:9	Mitochondrial
GmPHT3.4	LOC100800530	Glyma.08G008900	8	693228..696708	1068	XP_003532391.2	355	9.37	38.0	0.115	0	chlo:9	Mitochondrial
GmPHT3.5	LOC100788974	Glyma.11G087800	11	6610115..6614787	921	XP_003537697.1	306	9.22	33.7	0.186	1	nucl:5	Mitochondrial
GmPHT3.6	LOC547633	Glyma.16G050000	16	4788380..4801207	1119	NP_001235652.2	372	9.25	39.8	0.163	0	chlo:4	Mitochondrial
GmPHT3.7	LOC100801963	Glyma.16G050100	16	4798523..4800531	1020	XP_025981816.1	339	9.38	36.8	0.184	0	chlo:11	Mitochondrial
GmPHT3.8	LOC100804257	Glyma.16G146700	16	30755899..30759360	1050	XP_003548029.1	350	9.25	37.6	0.101	0	cyto:8	Mitochondrial
GmPHT3.9	LOC548006	Glyma.19G101100	19	34833393..34841869	1128	NP_001237304.1	375	9.35	39.8	0.193	0	chlo:4	Mitochondrial
GmPHT4.1	LOC100789297	Glyma.02G224200	2	41175313..41180091	1551	XP_003519242.1	516	10.01	55.7	0.479	12	plas:7	Membrane bound Chloroplast
GmPHT4.2	LOC100782221	Glyma.03G008200	3	793191..799880	1782	XP_003520968.1	593	9.36	65.5	0.220	10	plas:11	Membrane bound Chloroplast
GmPHT4.3	LOC100793618	Glyma.07G069600	7	6307073..6313498	1779	XP_003528848.1	592	9.37	65.2	0.218	10	plas:10	Membrane bound Chloroplast
GmPHT4.4	LOC100819182	Glyma.07G144700	7	17231556..17235971	1515	XP_003529140.1	504	9.88	55.0	0.509	11	chlo:6	Membrane bound Chloroplast
GmPHT4.5	LOC100812608	Glyma.07G274200	7	44592193..44600634	1290	NP_001239989.1	429	9.58	46.6	0.659	10	plas:8	Membrane bound Golgi
GmPHT4.6	LOC100787020	Glyma.11G175800	11	20331840..20340143	1554	XP_006591210.1	517	9.41	57.3	0.223	10	plas:9	Membrane bound Chloroplast
GmPHT4.7	LOC100806856	Glyma.13G162900	13	27823310..27833361	1587	XP_003541488.1	525	7.16	57.4	0.339	7	chlo:7	Membrane bound Chloroplast
GmPHT4.8	LOC100785142	Glyma.14G190900	14	45569552..45574890	1548	XP_003544870.1	515	9.93	55.8	0.540	11	plas:7	Membrane bound Chloroplast
GmPHT4.9	LOC100809973	Glyma.17G000300	17	17407..21108	1290	XP_003550165.1	429	9.68	46.5	0.624	10	plas:8	Membrane bound Golgi
GmPHT4.10	LOC100792616	Glyma.17G108300	17	8486013..8495685	1575	XP_003549724.1	524	6.83	57.5	0.330	7	plas:7	Membrane bound Chloroplast
GmPHT4.11	LOC100792104	Glyma.18G066000	18	6041786..6050082	1476	XP_003551308.1	491	9.09	53.8	0.411	10	plas:6	Membrane bound Chloroplast
GmPHT4.12	LOC100784401	Glyma.20G002000	20	213184..219283	1794	XP_003556597.1	597	9.12	65.7	0.295	10	plas:10	Membrane bound Chloroplast
GmPHT5.1	LOC100808181	Glyma.09G128500	9	32046109..32052575	2088	XP_003533972.1	695	6.17	78.2	0.214	11	plas:10	Membrane bound Vacuolar
GmPHT5.2	LOC100777530	Glyma.09G263400	9	48091634..48104775	2094	XP_003534584.1	697	6.14	78.6	0.209	10	plas:11	Membrane bound Vacuolar
GmPHT5.3	LOC100777032	Glyma.10G229900	10	46001071..46006711	2094	XP_014618833.1	697	6.22	78.7	0.170	11	plas:11	Membrane bound Vacuolar
GmPHT5.4	LOC100778938	Glyma.16G176100	16	33736100..33743654	2088	XP_003548146.1	695	6.36	78.2	0.200	11	plas:10	Membrane bound Vacuolar
GmPHT5.5	LOC100793709	Glyma.18G228700	18	51774933..51789151	2094	XP_006602776.1	697	6.55	78.7	0.232	11	plas:12	Membrane bound Vacuolar
GmPHT5.6	LOC100805436	Glyma.20G163400	20	40093249..40098816	2094	XP_003556130.1	697	6.52	78.9	0.158	11	plas:11	Membrane bound Vacuolar
GmPHO1.1	LOC100786083	Glyma.01G091800	1	27867550..27874181	2376	XP_003516868.1	791	9.14	92.2	-0.212	6	plas:8	Plasma membrane
GmPHO1.2	LOC100796500	Glyma.02G003700	2	431852..445384	2292	XP_006574509.1	763	9.24	88.4	-0.106	6	plas:10	Plasma membrane
GmPHO1.3	LOC100795093	Glyma.02G110600	2	10670899..10676578	2256	XP_006574913.1	751	8.85	88.5	-0.316	4	plas:8.5	Plasma membrane
GmPHO1.4	LOC100787164	Glyma.02G130200	2	13340016..13346701	2370	XP_003518826.1	789	9.12	92.0	-0.219	6	plas:8	Plasma membrane
GmPHO1.5	LOC100819185	Glyma.07G228400	7	40691412..40698177	2412	XP_006583960.1	803	9.45	93.4	-0.183	9	plas:12	Plasma membrane
GmPHO1.6	LOC100780548	Glyma.09G235200	9	45765397..45771516	2280	XP_006587738.1	759	9.28	87.7	-0.103	6	plas:11	Plasma membrane
GmPHO1.7	LOC100781658	Glyma.10G004800	10	457173..468529	2295	XP_006588543.1	764	9.41	88.4	-0.111	5	plas:9	Plasma membrane
GmPHO1.8	LOC100794580	Glyma.10G183300	10	41633437..41645279	2325	XP_006589283.1	774	9.06	90.1	-0.117	6	plas:10	Plasma membrane
GmPHO1.9	LOC100795104	Glyma.18G261900	18	54775710..54781535	2331	XP_003552542.1	776	9.24	89.3	-0.114	6	plas:9	Plasma membrane
GmPHO1.10	LOC100805979	Glyma.20G031700	20	3929374..3936931	2409	XP_006605562.1	802	9.27	93.0	-0.163	8	plas:12	Plasma membrane
GmPHO1.11	LOC100811310	Glyma.20G032400	20	4183317..4191520	2388	XP_003556776.1	795	9.38	92.8	-0.233	8	plas:12	Plasma membrane
GmPHO1.12	LOC100813281	Glyma.20G032500	20	4194291..4202575	2391	XP_003556778.1	796	9.32	92.6	-0.195	8	plas:11	Plasma membrane
GmPHO1.13	LOC100813818	Glyma.20G032600	20	4204879..4212818	2361	XP_003556779.1	786	9.24	91.7	-0.121	8	plas:12	Plasma membrane
GmPHO1.14	LOC100802251	Glyma.20G206900	20	44375656..44385721	2325	XP_006606372.1	774	9.14	89.9	-0.122	8	plas:9	Plasma membrane

Note: - indicates that the corresponding PHTs gene was not found in the NCBI soybean database. 1 represents the number of the GmPHT gene in the NCBI soybean database; 2 represents the number of the GmPHT gene in the Phytozome soybean database.

Collinearity analysis among members of the GmPHTs gene family. There are currently three ways in which gene is generated, including whole-genome duplication (polyploidy), large segmental duplication events and tandem duplication event ³⁸. Chromosomal distribution and Collinearity analysis of the GmPHTs gene（Fig. 2）shows that, 49% of the 57 GmPHTs gene family members were found to arise through large segmental duplication events and 14% through tandem duplication event. In the GmPHT1 subfamily, the GmPHT1.1 and GmPHT1.4, GmPHT1.2 and GmPHT1.11 genes are generated by a large segmental duplication event in the soybean chromosome; the GmPHT1.6 and GmPHT1.7, GmPHT1.13 and GmPHT1.14 genes are generated by tandem duplication event. two of the GmPHT2 members were formed by large segmental duplication events. GmPHT 3.1 and GmPHT3.5, GmPHT3.3 and GmPHT3.4, and GmPHT3.9 in the GmPHT3 subfamily have common ancestor genes with GmPHT3.6 and GmPHT3.7 that were generated by large segmental duplication events; ancestor genes of GmPHT3.6 and GmPHT3.7 genes Tandem duplications occurred. both the GmPHT4 and GmPHT5 subfamilies expanded the gene family size with only large segmental duplication events (GmPHT4.1 and GmPHT4.8, GmPHT4.5 and GmPHT4.9, GmPHT4.6 and GmPHT4.11 genes; GmPHT5.1 and GmPHT5:4, GmPHT5.3 and GmPHT5.6 genes). of the GmPHO1 subfamily, GmPHO1.1 and GmPHO1.4, GmPHO1.2 and GmPHO1.7, GmPHO1.8 and GmPHO1.14 genes were generated by large segmental duplication events, and GmPHO1.11, GmPHO1.12 and GmPHO1.13 genes were formed by tandem duplication event. It suggested that large segmental duplication also played a major driving force for GmPHTs evolution in addition to tandem duplication.

Gene structure and protein-specific motif analysis of the GmPHTs gene. From the perspective of gene structure, the GmPHO1 subfamily can be divided into 2 Groups, and Group I contain 8 genes including GmPHO1.11, GmPHO1.12 and GmPHO1.13. The three pairs of GmPHO1 genes form Group II, which differs from the Group I genes mainly in the length of the first exon. Members of the GmPHT3 subfamily have 5 or 6 exons, but the difference is mainly in whether the last exon is split or not. GmPHT4.1 and GmPHT4.8 both contain 8 exons, GmPHT4.5 and GmPHT4.9 genes contain only 1 exon, and GmPHT4.7 and GmPHT4.10 genes contain a maximum of 15 exons. The 14 members of the GmPHT1 subfamily can also be divided into two categories based on the results of gene structure, with the exception of GmPHT1.1 and GmPHT1.4, which have two exons, the other 10 genes in Group III have only one exon, and GmPHT1.3 and GmPHT1.12, which have similar structures and three exons. GmPHO1 subfamily has a maximum of 10 specific motifs and the number of motifs is the same for each member of the GmPHT3 and GmPHT5 subfamilies. The protein sequences of the GmPHT1 subfamily members contain 7-9 specific motifs, notably, the four gene pairs generated by large block repeats or tandem repeats and the GmPHT1.5 gene have exactly the same 9 motifs, whereas the protein encoded by the five genes generated by non-repetitive events has only 7 motifs except for 8 and 9. Only the number of specific motifs varied significantly among members of the GmPHT4 subfamily, ranging from 1 to 4 (Fig. 3).

Identification of the cis-acting regulatory elements. To understand the potential transcriptional regulation of GmPHTs, we performed the analysis based on the DNA sequences of the promoter regions. The 2.0-kb upstream region of the initiation codon as the promoter region of the gene to analyze which specific regulatory elements are present in the promoter region of each gene (Fig. 4, Table S2). The GmPHTs gene family has the most light-responsive elements, accounting for 43.26% of all promoter elements, and every gene has more or less of this type of promoter element. The next most abundant is the abiotic stress response element, which mainly responds to adverse environmental conditions such as heat, low temperature, anaerobic, drought, and low phosphorus. The phytohormone response elements mainly include abscisic acid, methyl jasmonate, gibberellin, ethylene, salicylic acid, and growth hormone response elements. Except for the GmPHT3.1 gene, the promoters of all genes had phytohormone-like response elements, differing only in type and number. Biological stress response elements were the least, accounting for only about 5%, and none of the members of the PHT2 subfamily had biological stress response elements. It is thus hypothesized that each member of the GmPHT genes family has an important role in the growth and development of soybean and resistance to various external growth-adverse stimuli. The basic biological function of PHT proteins has been reported to be the transport of inorganic phosphorus, and the application of phosphorus has a mitigating effect on drought ⁴⁰, so among the promoter elements, the low phosphorus response element P1BS and the drought-inducing element MBS are the focus of our attention. only 13 of the 57 genes have promoter regions containing both the low phosphorus response element P1BS and the drought-inducing element MBS. Therefore, further expression analysis was performed for 13 genes, Including GmPHT1.1, GmPHT1.7, GmPHT1.10, GmPHT1.14, GmPHT3.4, GmPHT3.5, GmPHT3.6, GmPHT4.7, GmPHT4.8, GmPHT4.10, GmPHO1.4, GmPHO1.5 and GmPHO1.7 were subjected to further analysis.

Differential expression profiles of the GmPHTs genes under low phosphorus and drought stress. So far, little is known about the specificity of tissues or organs of the soybean PHT genes, which may elucidate their functions in detail, because the GmPHTs family members in soybean have not been systemically examined. By the results of the preliminary qRT-PCR experiments (Data not shown) and the gene microarray data of soybean, GmPHT1.10, GmPHT1.14, GmPHT3.4 and GmPHT3.6 genes were not expressed among the 13 genes. Therefore, only the remaining nine genes were examined separately for their expression under simultaneous low phosphorus drought treatment conditions (Fig. 5). GmPHT1.1 gene was highly expressed in roots only, GmPHT3.5 gene was expressed in leaves only, GmPHT1.7, GmPHT4.7, GmPHT4.8, GmPHT4.10, GmPHO1.4, GmPHO1.5 and GmPHO1.7 genes were expressed in both roots and leaves. The GmPHT1.7 and GmPHT4.8 genes were expressed in both leaves and roots, and it was inferred that the GmPHT1.7 and GmPHT4.8 genes may play important roles in both roots and leaves, and the GmPHT1.1 gene acts synergistically in the roots during the seedling stage of soybean. In contrast, GmPHT4.10, GmPHO1.4, GmPHO1.5, and GmPHO1.7 genes were expressed in the roots but at low levels, and these three genes may not play a major role in the roots.

The expression pattern of these nine genes changed again when soybean was treated with both low phosphorus and drought for 12 h (Fig. 6). GmPHT3.5, GmPHT4.7, GmPHT4.10, GmPHO1.5, GmPHO1.7, and GmPHT1.7 was significantly reduced. gene expression of GmPHT1.1 in the roots increased gradually with time, and its expression increased significantly when treated with low phosphorus and drought for 12 h. gradually increased. The expression pattern of GmPHO1.4 gene in leaves was similar to that of GmPHT1.1 gene, both of which decreased first and then showed a significant increase with time. Only the expression of GmPHT4.8 gene in leaves was not affected by low phosphorus and drought, and the expression was basically the same as that of the control, but the expression in roots was significantly lower.

Coexpression networks of the PHTs family genes in soybean. To further unravel the coexpression relationships between PHTs family genes and other genes, we calculated the interaction weight values of the target gene sets based on the FPKM values from the RNA-seq data. Fig. 7 shows all the genes interacting with GmPHTs genes. In general, among the interacting genes, GmPHT1:1 interacted with 31 genes, while GmPHO1:4 interacted with 57 genes. Three genes interacted most strongly with GmPHT1:1, and six genes were co-expressed with GmPHO1:4. No other genes were found that interacted strongly with other PHTs genes under low phosphorus stress (Fig 7). Among of them, included ubiquitin family genes, ubiquitin-conjugating enzyme genes. Ubiquitination modifications play a central regulatory role in the response to low phosphorus stress in plants, may be involved in regulating the absorption of elemental phosphorus.

Fig 7 Coexpression networks of 2 PHTs family genes in soybean. The size of the node is proportional to the degree of the node, the more edges connected to the node, the greater its degree and the larger the node; the color indicates the strength of the interaction, the redder the color the stronger the interaction. The PHTs family genes located in the center of the network, while the most coexpressed genes were displayed in each network.

As an essential, nonsubstitutable element for plant growth, P plays a wide range of structural and biological roles ⁴¹. P deficiency influences numerous biological processes in crop plants, such as rice, soybean and wheat, markedly reducing crop yield ^42–45. Plants need the phosphorus they need from the soil, since soil phosphorus concentration is lower than that of plant tissues ⁴⁶, This sharp concentration gradient between the plant and the soil indicates the indispensable roles of PHTs, which can regulate Pi absorption.

Pi transporters currently identified in Arabidopsis, rice, soybean, maize, wheat, poplar, Medicago truncatula, and the Halophyte E. salsugineum ⁴⁷. The concerted action of Pi transporters ensures Pi acquisition and distribution among tissues and cytosolic Pi homeostasis ⁴⁵. Therefore, engineered alterations of the expression of Pi transporters provide an opportunity to optimize uptake and distribution of Pi in crops to improve yield ⁴⁸. A systematic analysis of the whole PHT gene family in soybean was conducted in this study, which will provide an opportunity to find candidate genes that play roles in phosphate use efficiency and abiotic stress response. A total of 57 PHT genes are identified in soybean and show quite high similarity to their corresponding members of the PHT gene family in Arabidopsis (Fig. 1). This implies that these PHT genes are evolutionarily conserved and functionally similar in soybean and Arabidopsis.

Gene duplication is considered one of the primary driving forces in the evolution of genomes and genetic systems. Of all the duplication patterns, segmental and tandem duplications have been purported to be the two main causes of gene family expansion in plants ⁴⁹. In the current research, 29 of the 57 GmPHTs (87.8%) in the soybean genome were associated with large segmental duplication events, Moreover, only 7 tandem duplication events were identified (Fig. 2). Taken together, our results indicate that large blockbuster duplication and tandem duplication events are the main force for the expansion of the PHT gene family in soybean. A relatively large number of members within a family suggests successful expansion and rearrangement of the genome by extensive duplication that occurred frequently during evolution ⁵⁰.

Cis elements may control the efficiency of promoters and thus regulate the expression of the genes that they control by interacting with the corresponding trans-regulatory factors ⁵¹. Studies of cis elements might play a crucial role in dissecting the functions of genes. But the cis elements of all PHT gene family members in soybean have not yet been identified. In this study, the upstream regions of all the GmPHTs contain cis elements affecting light, hormone, defense and tissue-specific expression. These findings suggest that the expression of these GmPHT genes might be regulated by many factors, such as light-, hormone-, and defense-related factors. The presence of these cis elements in soybean GmPHT genes and their likely roles in regulating gene expression suggest that GmPHT genes may be involved in various stress responses in soybean Some cis elements function under phosphate deficiency, for example, the P1BS element can be bound to PHR1 (a MYB transcription factor) to potentially regulate the phosphate starvation response in Arabidopsis ⁵², PHR1 has been suggested as a central integrator to role in the transcriptional regulation of phosphate starvation responses ⁵³. In the current study, P1BS-like elements were found in 13 of the 57 GmPHT promoters examined suggesting that expression of these genes might be influenced by Pi starvation. Similarly, P1BS-like elements have also been detected in OsPHT genes in rice ⁵¹. These elements are found to be in the promoters of phosphate regulated genes, not only in Arabidopsis but also in other plant species such as barley and rice ^{51, 54, 55}, which suggests that they may participate in a conserved signaling pathway for the phosphate starvation response in plants. The existence of P1BS-like elements in soybean, rice, and Arabidopsis suggests that they may serve a similar role in different species.

Drought and Pi deficiency stress often interfere with plant metabolism via various stress signals and hormonal changes that play essential roles in regulation processes under natural conditions ⁵⁶. So far, several studies have found that Pi transporters are involved in response to drought stress. Recently, several genes encoding Pi transporters from poplar were found to be regulated by drought stress, these genes especially those up-regulated by drought stress at low Pi level may contribute to drought tolerance of poplar plants in Pi-limited soils ⁵⁷. Secondly, using drought inducible promoters to control the expression of Pi transporters or their regulatory factors may be a potential strategy to improve crop PUE under drought stress conditions. Therefore, engineered alterations of the expression of Pi transporters provide an opportunity to optimize uptake and distribution of Pi in crops to improve yield. In this study, the expression patterns of GmPHT genes were analyzed under low phosphorus, drought and simultaneous low phosphorus drought stress conditions, and the results showed that the expression patterns of the genes differed under different treatment conditions. These results suggest that of crosstalk occurs among the P-starvation response and salt and drought stress responses, and Pi uptake in plants likely changes in association with the altered expression of the PHT1 genes under drought and salt stresses⁴⁸.Our results shown that under simultaneous drought and low phosphorus treatments, the expression of GmPHT1.7 and GmPHT1.1 genes in roots significantly increased, while the expression of GmPHO1.4 gene in leaves showed a significant increase with the continuation of treatment time, and the expression of this gene in roots significantly decreased. The expression of GmPHT4.8 gene in leaves was not affected by low phosphorus and drought, but the expression in roots was significantly reduced (Fig. 6). The identification of these genes is therefore of great significance for the development of transgenic crops to cope with low phosphorus and drought stress.

Plant Pi transporters play an essential role in Pi acquisition and distribution. The family of phosphate transporters (PHTs) mediates the uptake and translocation of Pi inside the plants. In this study, a total of 57 PHT-domain-containing protein genes were identified in soybean genome, (GmPHT1, GmPHT2, GmPHT3, GmPHT4, GmPHT5 and GmPHO1) had been found. Only 13 of the 57 genes have promoter regions containing both the low phosphorus response element P1BS and the drought-inducing element MBS, 4 genes were not expressed among the 13 genes. Therefore, only the remaining nine genes were examined separately for their expression under simultaneous low phosphorus drought treatment conditions. The results of expression analysis showed that the 9 PHT-domain-containing protein genes displayed various expression patterns. The above results implied that they may play different roles in phosphate nutrition of Soybean different tissues and development stages. These results provide references for the further study of Soybean PHT-domain-containing proteins family genes. Moreover, this study provides the selection of candidate genes for functional research and genome editing in Soybean phosphate nutrition.

Identification of the GmPHT family genes in soybean. The protein sequences of the AtPHT genes family were used for TBlastN alignment in the soybean genome database Phytozome (v12.1) to obtain sequences similar to members of the AtPHT genes family. The obtained sequences were then analyzed in the NCBI CDD database (v3.15) for conserved structural domains to identify whether the obtained similar sequences were GmPHTs proteins. The sequence of the protein finally identified as GmPHTs was sequence corrected in the NCBI soybean database. Data on the isoelectric point, molecular weight, and hydrophilicity of the proteins were obtained using the ProtParam tool in the ExPASy (https://web.expasy.org/) online analysis website.

Phylogenetic, Multiple alignment and protein motif analysis. The PHT protein sequences of Arabidopsis and soybean were selected for multiple sequence alignment using the ClustalW program in BioEdit (v7.0.5) software. The PHT protein sequences of yeast were used as outgroup to construct a phylogenetic tree by MEGA (v7.0.26) software. The method used was the Jones-Taylor-Thornton (JTT) model, and Bootstrap was 1000 replicates.

The PLAZA website (https://bioinformatics.psb.ugent.be/plaza) was used to analyze the collinearity of the soybean genome and finally to obtain the regions where large segments of duplication occurred in the soybean genome.

The number of introns and exons of genes was analyzed and counted using the online analysis website GSDS (v2.0) (http://gsds.cbi.pku.edu.cn). The unique or shared motifs in the proteins were done using the MEME line analysis website (http://meme-suite.org/).

Regulatory promoter element analysis in GmPHT genes. The upstream sequence of the first 2000 bp of the gene obtained from the soybean genome database phytozome (V12.1) was taken as the promoter region of the gene. The promoter element prediction was performed for all GmPHT members using the promoter online prediction website PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The promoter elements other than the basic promoter elements (TATA box and CAAT box) were mapped using the GSDS online mapping tool for promoter elements.

Plant materials and growth condition. The experiments were conducted with the main cultivar Changnong 26 in Jilin Province (Validation No.: JI Audited Bean 2010004). To identify tissue-preferentially expressed genes, seedlings of Chang Nong 26 were grown under normal conditions and cultured in sand culture at 25°C with 50% humidity and 13 h of light and 9 h of darkness, using 1/4 Hoagland (containing 0.5 mM KH₂PO₄) as the culture medium to provide essential nutrients for the soybean. The plants were converted to hydroponic culture when they reached the three-leaf stage, and the culture medium was still 1/4 Hoagland containing 0.5 mM KH₂PO₄. After 12 h of hydroponic culture, the plants were transferred to 1/4 Hoagland culture with 0.01 mM KH₂PO₄, 1/4 Hoagland culture with 2% PEG8000 (containing 0.5 mM KH₂PO₄) and 1/4 Hoagland culture with 2% PEG8000 containing 0.01 mM KH₂PO₄ for Hydroponic culture. Three treatments were set up with three replications, continuous normal phosphorus treatment, continuous low phosphorus, and stressed low phosphorus treatment. Samples were taken on time for 12h in hydroponics, and the roots and leaves were stored separately at -80°C for backup.

Coexpression networks of the GmPHT family genes using RNA-seq data. Gene coexpression network analysis was performed based on the RNA-seq data. For the RNA-seq experiment, fully expanded leaves and roots were taken when the plants were treated with low phosphorus nutrient solution until to 3 leaves separately for RNA extraction with three biological replicates. The transcript abundance (FPKM value) of each gene was calculated based on the length of the gene and the reads mapped to that gene. The interactions of the target gene sets were retrieved from the STRING protein database (http://string-db.org/), and the weight value of the target gene sets was calculated using the WGCNA R package based on the FPKM values. The gene coexpression networks were visualized by Cytoscape software ⁵⁸.

Quantitative real-time PCR analysis. RNA was extracted using Trizol (TAKARA, Beijing) and reverse transcribed using PrimeScriptTM RT reagent Kit with gDNA Eraser kit (TAKARA, Beijing). Fluorescent quantitative PCR primers were designed according to the specific sequences of each gene, see Table S1. Letin was used as the internal reference gene, and the fluorescent quantitative PCR was performed using the TAKARA SYBR Premix Ex Taq II kit (TAKARA, Beijing, China) with an ABI StepOne Plus real-time fluorescent quantitative PCR instrument (ABI, USA, model 7300). 25 µl of the reaction system contained 1 µl cDNA, 10 µl SYBR The PCR program was: 95°C for 10 min (pre-denaturation), 95°C for 3 s (denaturation) and 60°C for 1 min (annealing-extension) for 40 cycles. The lysis curve procedure was: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Data were calculated using the 2^−ΔΔct algorithm for relative expression ⁵⁹. All experiments were performed in three replicates. Statistix (v 8.1) software was used to analyze the significance of the data.

Statistical Analysis Of Data

Statistics was performed by Duncan’s test or Student’s t test. Significance of differences was defined as *P < 0.05, ** P < 0.01.

Acknowledgements

This study was supported by the following funding sources: Jilin Science and technology development plan project (20210101045JC, 20200403016SF). Thirteenth Five-Year Plan" Science and Technology Project of Jilin Provincial Education Department (JJKH20200335KJ).

AUTHOR CONTRIBUTIONS

XY. and M-Y Y. designed and supervised the project, X-SW. and Z-H W analyzed the data, and wrote the manuscript, YF, R-J Y, and LW participated in the material preparation, PT, S-Y L revised the manuscript, all authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing interests.

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/Supplementary Material. The Supplementary Material for this article can be found online at: https://www.ncbi.nlm.nih.gov/sra/PRJNA871448. Accession number：PRJNA871448, NCBI SRA database.

ACKNOWLEDGMENTS

We thank to researcher of Soybean Research Institute of Changchun Academy of Agricultural Sciences for providing experimental materials

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Comprehensive Sequence and Expression Profile Analysis of the Phosphate Transporter Gene Family in Soybean

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