Mining and utilization of salinity tolerant legumes in tropical coastal agroecosystems: An overview

Yiming Liu; Mary Atieno; Juan Andres Cardoso; Hubiao Yang; Bin Xu; Rongshu Dong; Linling Yan; Chunqiong Huang; Hengfu Huan; Daogeng Yu; Sabine Douxchamps; Guodao Liu; Yiming Liu; Mary Atieno; Juan Andres Cardoso; Hubiao Yang; Bin Xu; Rongshu Dong; Linling Yan; Chunqiong Huang; Hengfu Huan; Daogeng Yu; Sabine Douxchamps; Guodao Liu

doi:10.48130/GR-2022-0010

2022 Volume 2

Article Contents

Abstract

Introduction

Materials and methods

Identification of aqp genes through genome-wide

Phylogenetic analysis and classification of aqp genes

Analysis of the npa motif and transmembrane domains

Characterization of aqp genes and protein properties

In silico tissue expression profiling of aqps genes

Plant material and seed treatment with different substances

Rna-seq of imbibing embryos and data analysis

Quantitative real-time pcr (qrt-pcr )

Results

Identification and classification of the aqp genes in garden pea

Genome distribution and gene structure analysis of pea aqps

Characterization of the npa motifs

Predicted subcellular localization of psaqps

Tissue expression profiles of psaqps

Psaqps expressions in response to osmotic stress and fullerol treatment in imbibing embryos

Validation of the degs through qrt-pcr

Discussion

Conclusions

Deposition of raw data

Acknowledgments

Conflict of interest

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References

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Mining and utilization of salinity tolerant legumes in tropical coastal agroecosystems: An overview

1.
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS) / Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture and Rural Affairs, Danzhou 571737, Hainan, China
2.
Tropical Forages Program, Alliance Bioversity-CIAT, Hanoi 100000, Vietnam
3.
International Center for Tropical Agriculture (CIAT), Cali 763537, Colombia
4.
College of Agro-grassland Science, Nanjing Agricultural University, Nanjing 210095, China
^# These authors contributed equally: Yiming Liu, Mary Atieno

More Information

Corresponding authors: S.Douxchamps@CGIAR.ORG; liuguodao2008@163.com

Received: 18 September 2022
Accepted: 16 November 2022
Published online: 21 December 2022
Grass Research 2, Article number: 10 (2022) | Cite this article

Abstract

Coastal saline soils are increasing year by year caused by climate change and human activities. Most of the coastal saline soils are idle due to their high salinity level and few crops can grow normally. Salinity tolerant legumes are naturally tolerant to salt, which can ecologically cover the coastal saline soil, enhance soil fertility by symbiotic nitrogen fixation and increase the smallholder farmers’ benefits in terms of forage, green manure, food or medicine. However, few reports are available for the systematic evaluation of salinity tolerant legumes. This review summarizes and evaluates currently available salinity tolerant legume species that could potentially be used and discusses their potential for integration into smallholder mixed coastal systems of the Asia-Pacific region. Fourty four salinity tolerant legumes were summarized, six of them showed a high level of salinity tolerance, 17 of them showed a moderate level of salinity tolerance and 21 of them showed potential salinity tolerance but need to be further studied. Many gaps such as combined tolerance evaluation, nitrogen fixation efficiency, animal feeding experiments and salinity tolerant rhizobia screening/inoculants exist. Case studies demonstrate legumes could be used to reclaim coastal saline soils, but commitment and support from government and public services are necessary to address both seed system and extension needs, through the provision of adequate incentives, policies and development efforts.
- Forage legume,
- Rhizobia,
- Salinity tolerance,
- Intercropping system,
- Coastal smallholder farmers.

INTRODUCTION

Aquaporin’s (AQPs) are small (21–34 kD) channel-forming, water-transporting trans-membrane proteins which are known as membrane intrinsic proteins (MIPs) conspicuously present across all kingdoms of life. In addition to transporting water, plant AQPs act to transport other small molecules including ammonia, carbon dioxide, glycerol, formamide, hydrogen peroxide, nitric acid, and some metalloids such as boron and silicon from the soil to different parts of the plant^[1]. AQPs are typically composed of six or fewer transmembrane helices (TMHs) coupled by five loops (A to E) and cytosolic N- and C-termini, which are highly conserved across taxa^[2]. Asparagine-Proline-Alanine (NPA) boxes and makeup helices found in loops B (cytosolic) and E (non-cytosolic) fold back into the protein's core to form one of the pore's two primary constrictions, the NPA region^[1]. A second filter zone exists at the pore's non-cytosolic end, where it is called the aromatic/arginine (ar/R) constriction. The substrate selectivity of AQPs is controlled by the amino acid residues of the NPA and ar/R filters as well as other elements of the channel^[1].

To date, the AQP gene families have been extensively explored in the model as well as crop plants^[3−9]. In seed plants, AQP distributed into five subfamilies based on subcellular localization and sequence similarities: the plasma membrane intrinsic proteins (PIPs; subgroups PIP1 and PIP2), the tonoplast intrinsic proteins (TIPs; TIP1-TIP5), the nodulin26-like intrinsic proteins (NIPs; NIP1-NIP5), the small basic intrinsic proteins (SIPs; SIP1-SIP2) and the uncategorized intrinsic proteins (XIPs; XIP1-XIP3)^[2,10]. Among them, TIPs and PIPs are the most abundant and play a central role in facilitating water transport. SIPs are mostly found in the endoplasmic reticulum (ER)^[11], whereas NIPs homologous to GmNod26 are localized in the peribacteroid membrane^[12].

Several studies reported that the activity of AQPs is regulated by various developmental and environmental factors, through which water fluxes are controlled^[13]. AQPs are found in all organs such as leaves, roots, stems, flowers, fruits, and seeds^[14,15]. According to earlier studies, increased AQP expression in transgenic plants can improve the plants' tolerance to stresses^[16,17]. Increased root water flow caused by upregulation of root aquaporin expression may prevent transpiration^[18,19]. Overexpression of Tamarix hispida ThPIP2:5 improved osmotic stress tolerance in Arabidopsis and Tamarix plants^[20]. Transgenic tomatoes having apple MdPIP1;3 ectopically expressed produced larger fruit and improved drought tolerance^[21]. Plants over-expressing heterologous AQPs, on the other hand, showed negative effects on stress tolerance in many cases. Overexpression of GsTIP2;1 from G. soja in Arabidopsis plants exhibited lower resistance against salt and drought stress^[22].

A few recent studies have started to establish a link between AQPs and nanobiology, a research field that has been accelerating in the past decade due to the recognition that many nano-substances including carbon-based materials are valuable in a wide range of agricultural, industrial, and biomedical activities^[23]. Carbon nanotubes (CNTs) were found to improve water absorption and retention and thus enhance seed germination in tomatoes^[24,25]. Ali et al.^[26] reported that Carbon nanoparticles (CTNs) and osmotic stress utilize separate processes for AQP gating. Despite lacking solid evidence, it is assumed that CNTs regulate the aquaporin (AQPs) in the seed coats^[26]. Another highly noticed carbon-nano-molecule, the fullerenes, is a group of allotropic forms of carbon consisting of pure carbon atoms^[27]. Fullerenes and their derivatives, in particular the water-soluble fullerols [C₆₀(OH)₂₀], are known to be powerful antioxidants, whose biological activity has been reduced to the accumulation of superoxide and hydroxyl^[28,29]. Fullerene/fullerols at low concentrations were reported to enhance seed germination, photosynthesis, root growth, fruit yield, and salt tolerance in various plants such as bitter melon and barley^[30−32]. In contrast, some studies also reported the phytotoxic effect of fullerene/fullerols^[33,34]. It remains unknown if exogenous fullerene/fullerol has any impact on the expression or activity of AQPs in the cell.

Garden pea (P. sativum) is a cool-season crop grown worldwide; depending on the location, planting may occur from winter until early summer. Drought stress in garden pea mainly affects the flowering and pod filling which harm their yield. In the current study, we performed a genome-wide identification and characterization of the AQP genes in garden pea (P. sativum), the fourth largest legume crop worldwide with a large complex genome (~4.5 Gb) that was recently decoded^[35]. In particular, we disclose, for the first time to our best knowledge, that the transcriptional regulations of AQPs by osmotic stress in imbibing pea seeds were altered by fullerol supplement, which provides novel insight into the interaction between plant AQPs, osmotic stress, and the carbon nano-substances.

MATERIALS AND METHODS

Identification of AQP genes through Genome-wide

The whole-genome sequence of garden pea ('Caméor') was retrieved from the URGI Database (https://urgi.versailles.inra.fr/Species/Pisum). Protein sequences of AQPs from two model crops (Rice and Arabidopsis) and five other legumes (Soybean, Chickpea, Common bean, Medicago, and Peanut) were used to identify homologous AQPs from the garden pea genome (Supplemental Table S1). These protein sequences, built as a local database, were then BLASTp searched against the pea genome with an E-value cutoff of 10⁻⁵ and hit a score cutoff of 100 to identify AQP orthologs. The putative AQP sequences of pea were additionally validated to confirm the nature of MIP (Supplemental Table S2) and transmembrane helical domains through TMHMM (www.cbs.dtu.dk/services/TMHMM/).

Phylogenetic analysis and classification of AQP genes

Further phylogenetic analysis was performed to categorize the AQPs into subfamilies. The pea AQP amino acid sequences, along with those from Medicago, a cool-season model legume phylogenetically close to pea, were aligned through ClustalW2 software (www.ebi.ac.uk/Tools/msa/clustalw2) to assign protein names. The unaligned AQP sequences to Medicago counterparts were once again aligned with the AQP sequences of Arabidopsis, rice, and soybean. Based on the LG model, unrooted phylogenetic trees were generated via MEGA7 and the neighbor-joining method^[36], and the specific name of each AQP gene was assigned based on its position in the phylogenetic tree.

Analysis of the NPA motif and transmembrane domains

By using the conserved domain database (CDD, www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), the NPA motifs were identified from the pea AQP protein sequences^[37]. The software TMHMM (www.cbs. dtu.dk/services/TMHMM/)^[38] was used to identify the protein transmembrane domains. To determine whether there were any alterations or total deletion, the transmembrane domains were carefully examined.

Characterization of AQP genes and protein properties

Basic molecular properties including amino acid composition, relative molecular weight (MW), and instability index were investigated through the online tool ProtParam (https://web.expasy.org/protparam/). The isoelectric points (pI) were estimated by sequence Manipulation Suite version 2 (www.bioinformatics.org/sms2)^[39]. The subcellular localization of AQP proteins was predicted using Plant-mPLoc^[40] and WoLF PSORT (www.genscript.com/wolf-psort.html)^{[ 41]} algorithms.

The gene structure (intron-exon organization) of AQPs was examined through GSDS ver 2.0^[42]. The chromosomal distribution of the AQP genes was illustrated by the software MapInspect (http://mapinspect.software.informer.com) in the form of a physical map.

In silico tissue expression profiling of AQPs genes

To explore the tissue expression patterns of pea AQP genes, existing NGS data from 18 different libraries covering a wide range of tissue, developmental stage, and growth condition of the variety ‘Caméor’ were downloaded from GenBank (www.ncbi.nlm.nih.gov/bioproject/267198). The expression levels of the AQP genes in each tissue and growth stage/condition were represented by the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) values. Heatmaps of AQPs gene were generated through Morpheus software (https://software.broadinstitute.org/morpheus/#).

Plant material and seed treatment with different substances

Different solutions, which were water (W), 0.3 M mannitol (M), and fullerol of different concentrations dissolved in 0.3 M mannitol (MF), were used in this study. MF solutions with the fullerol concentration of 10, 50, 100, and 500 mg/L were denoted as MF1, MF2, MF3, and MF4, respectively. Seeds of 'SQ-1', a Chinese landrace accession of a pea, were germinated in two layers of filter paper with 30 mL of each solution in Petri dishes (12 cm in diameter) each solution, and the visual phenotype and radicle lengths of 150 seeds for each treatment were analyzed 72 h after soaking. The radicle lengths were measured using a ruler. Multiple comparisons for each treatment were performed using the SSR-Test method with the software SPSS 20.0 (IBM SPSS Statistics, Armonk, NY, USA).

RNA-Seq of imbibing embryos and data analysis

Total RNA was extracted from imbibing embryos after 12 h of seed soaking in the W, M, and MF3 solution, respectively, by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The quality and quantity of the total RNA were measured through electrophoresis on 1% agarose gel and an Agilent 2100 Bioanalyzer respectively (Agilent Technologies, Santa Rosa, USA). The TruSeq RNA Sample Preparation Kit was utilized to construct an RNA-Seq library from 5 µg of total RNA from each sample according to the manufacturer's instruction (Illumina, San Diego, CA, USA). Next-generation sequencing of nine libraries were performed through Novaseq 6000 platform (Illumina, San Diego, CA, USA).

First of all, by using SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) the raw RNA-Seq reads were filtered and trimmed with default parameters. After filtering, high-quality reads were mapped onto the pea reference genome (https://urgi.versailles.inra.fr/Species/Pisum) by using TopHat (V2.1.0)^[43]. Using Cufflinks, the number of mapped reads from each sample was determined and normalised to FPKM for each predicted transcript (v2.2.1). Pairwise comparisons were made between W vs M and W vs M+F treatments. The DEGs with a fold change ≥ 1.5 and false discovery rate (FDR) adjusted p-values ≤ 0.05 were identified by using Cuffdiff^[44].

Quantitative real-time PCR (qRT-PCR )

qPCR was performed by using TOROGGreen® qPCR Master Mix (Toroivd, Shanghai, China) on a qTOWER®3 Real-Time PCR detection system (Analytik Jena, Germany). The reactions were performed at 95 °C for 60 s, followed by 42 cycles of 95 °C for 10 s and 60 °C for 30 s. Quantification of relative expression level was achieved by normalization against the transcripts of the housekeeping genes β-tubulin according to Kreplak et al.^[35]. The primer sequences for reference and target genes used are listed in Supplemental Table S3.

RESULTS

Identification and classification of the AQP genes in garden pea

The homology-based analysis identifies 41 putative AQPs in the garden pea genome. Among them, all but two genes (Psat0s3550g0040.1, Psat0s2987g0040.1) encode full-length aquaporin-like sequences (Table 1). The conserved protein domain analysis later validated all of the expected AQPs (Supplemental Table S2). To systematically classify these genes and elucidate their relationship with the AQPs from other plants' a phylogenetic tree was created. It clearly showed that the AQPs from pea and its close relative M. truncatula formed four distinct clusters, which represented the different subfamilies of AQPs i.e. TIPs, PIPs, NIPs, and SIPs (Fig. 1a). However, out of the 41 identified pea AQPs, 4 AQPs couldn't be tightly aligned with the Medicago AQPs and thus were put to a new phylogenetic tree constructed with AQPs from rice, Arabidopsis, and soybean. This additional analysis assigned one of the 4 AQPs to the XIP subfamily and the rest three to the TIP or NIP subfamilies (Fig. 1b). Therefore, it is concluded that the 41 PsAQPs comprise 11 PsTIPs, 15 PsNIPs, 9 PsPIPs, 5 PsSIPs, and 1 PsXIP (Table 2). The PsPIPs formed two major subgroups namely PIP1s and PIP2s, which comprise three and six members, respectively (Table 1). The PsTIPs formed two major subgroups TIPs 1 (PsTIP1-1, PsTIP1-3, PsTIP1-4, PsTIP1-7) and TIPs 2 (PsTIP2-1, PsTIP2-2, PsTIP2-3, PsTIP2-6) each having four members (Table 2). Detailed information such as gene/protein names, accession numbers, the length of deduced polypeptides, and protein structural features are presented in Tables 1 & 2

Table 1. Description and distribution of aquaporin genes identified in the garden pea genome.

Chromosome
S. No	Gene Name	Gene ID	Gene length (bp)	Location	Start	End	Transcription length (bp)	CDS length (bp)	Protein length (aa)
1	PsPIP1-1	Psat5g128840.3	2507	chr5LG3	231,127,859	231,130,365	675	675	225
2	PsPIP1-2	Psat2g034560.1	1963	chr2LG1	49,355,958	49,357,920	870	870	290
3	PsPIP1-4	Psat2g182480.1	1211	chr2LG1	421,647,518	421,648,728	864	864	288
4	PsPIP2-1	Psat6g183960.1	3314	chr6LG2	369,699,084	369,702,397	864	864	288
5	PsPIP2-2-1	Psat4g051960.1	1223	chr4LG4	86,037,446	86,038,668	585	585	195
6	PsPIP2-2-2	Psat5g279360.2	2556	chr5LG3	543,477,849	543,480,404	2555	789	263
7	PsPIP2-3	Psat7g228600.2	2331	chr7LG7	458,647,213	458,649,543	2330	672	224
8	PsPIP2-4	Psat3g045080.1	1786	chr3LG5	100,017,377	100,019,162	864	864	288
9	PsPIP2-5	Psat0s3550g0040.1	1709	scaffold03550	20,929	22,637	1191	1191	397
10	PsTIP1-1	Psat3g040640.1	2021	chr3LG5	89,426,473	89,428,493	753	753	251
11	PsTIP1-3	Psat3g184440.1	2003	chr3LG5	393,920,756	393,922,758	759	759	253
12	PsTIP1-4	Psat7g219600.1	2083	chr7LG7	441,691,937	441,694,019	759	759	253
13	PsTIP1-7	Psat6g236600.1	1880	chr6LG2	471,659,417	471,661,296	762	762	254
14	PsTIP2-1	Psat1g005320.1	1598	chr1LG6	7,864,810	7,866,407	750	750	250
15	PsTIP2-2	Psat4g198360.1	1868	chr4LG4	407,970,525	407,972,392	750	750	250
16	PsTIP2-3	Psat1g118120.1	2665	chr1LG6	230,725,833	230,728,497	768	768	256
17	PsTIP2-6	Psat2g177040.1	1658	chr2LG1	416,640,482	416,642,139	750	750	250
18	PsTIP3-2	Psat6g054400.1	1332	chr6LG2	54,878,003	54,879,334	780	780	260
19	PsTIP4-1	Psat6g037720.2	1689	chr6LG2	30,753,624	30,755,312	1688	624	208
20	PsTIP5-1	Psat7g157600.1	1695	chr7LG7	299,716,873	299,718,567	762	762	254
21	PsNIP1-1	Psat1g195040.2	1864	chr1LG6	346,593,853	346,595,716	1863	645	215
22	PsNIP1-3	Psat1g195800.1	1200	chr1LG6	347,120,121	347,121,335	819	819	273
23	PsNIP1-5	Psat7g067480.1	2365	chr7LG7	109,420,633	109,422,997	828	828	276
24	PsNIP1-6	Psat7g067360.1	2250	chr7LG7	109,270,462	109,272,711	813	813	271
25	PsNIP1-7	Psat1g193240.1	1452	chr1LG6	344,622,606	344,624,057	831	831	277
26	PsNIP2-1-2	Psat3g197520.1	669	chr3LG5	420,092,382	420,093,050	345	345	115
27	PsNIP2-2-2	Psat3g197560.1	716	chr3LG5	420,103,168	420,103,883	486	486	162
28	PsNIP3-1	Psat2g072000.1	1414	chr2LG1	133,902,470	133,903,883	798	798	266
29	PsNIP4-1	Psat7g126440.1	1849	chr7LG7	209,087,362	209,089,210	828	828	276
30	PsNIP4-2	Psat5g230920.1	1436	chr5LG3	463,340,575	463,342,010	825	825	275
31	PsNIP5-1	Psat6g190560.1	1563	chr6LG2	383,057,323	383,058,885	867	867	289
32	PsNIP6-1	Psat5g304760.4	5093	chr5LG3	573,714,868	573,719,960	5092	486	162
33	PsNIP6-2	Psat7g036680.1	2186	chr7LG7	61,445,341	61,447,134	762	762	254
34	PsNIP6-3	Psat7g259640.1	2339	chr7LG7	488,047,315	488,049,653	918	918	306
35	PsNIP7-1	Psat6g134160.2	4050	chr6LG2	260,615,019	260,619,068	4049	1509	503
36	PsSIP1-1	Psat3g091120.1	3513	chr3LG5	187,012,329	187,015,841	738	738	246
37	PsSIP1-2	Psat1g096840.1	3609	chr1LG6	167,126,599	167,130,207	744	744	248
38	PsSIP1-3	Psat7g203280.1	2069	chr7LG7	401,302,247	401,304,315	720	720	240
39	PsSIP2-1-1	Psat0s2987g0040.1	706	scaffold02987	177,538	178,243	621	621	207
40	PsSIP2-1-2	Psat3g082760.1	3135	chr3LG5	173,720,100	173,723,234	720	720	240
41	PsXIP2-1	Psat7g178080.1	2077	chr7LG7	335,167,251	335,169,327	942	942	314
bp: base pair, aa: amino acid.

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Figure 1. Phylogenetic analysis of the identified AQPs from pea genome. (a) The pea AQPs proteins aligned with those from the cool-season legume Medicago truncatual. (b) The four un-assigned pea AQPs in (a) (denoted as NA) were further aligned with the AQPs of rice, soybean, and Arabidopsis by using the Clustal W program implemented in MEGA 7 software. The nomenclature of PsAQPs was based on homology with the identified aquaporins that were clustered together.

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Table 2. Protein information, conserved amino acid residues, trans-membrane domains, selectivity filter, and predicted subcellular localization of the 39 full-length pea aquaporins.

S. No	AQPs	Gene	Length	TMH	NPA	NPA	ar/R selectivity filter				pI	WoLF PSORT	Plant-mPLoc
S. No	AQPs	Gene	Length	TMH	LB	LE	H2	H5	LE1	LE2	pI	WoLF PSORT	Plant-mPLoc
Plasma membrane intrinsic proteins (PIPs)
1	PsPIP1-1	Psat5g128840.3	225	4	NPA	0	F	0	0	0	8.11	Plas	Plas
2	PsPIP1-2	Psat2g034560.1	290	2	NPA	NPA	F	H	T	R	9.31	Plas	Plas
3	PsPIP1-4	Psat2g182480.1	288	6	NPA	NPA	F	H	T	R	9.29	Plas	Plas
4	PsPIP2-1	Psat6g183960.1	288	6	NPA	NPA	F	H	T	0	8.74	Plas	Plas
5	PsPIP2-2-1	Psat4g051960.1	195	3	0	0	F	H	T	R	8.88	Plas	Plas
6	PsPIP2-2-2	Psat5g279360.2	263	5	NPA	NPA	F	H	T	R	5.71	Plas	Plas
7	PsPIP2-3	Psat7g228600.2	224	4	NPA	0	F	F	0	0	6.92	Plas	Plas
8	PsPIP2-4	Psat3g045080.1	288	6	NPA	NPA	F	H	T	R	8.29	Plas	Plas
Tonoplast intrinsic proteins (TIPs)
1	PsTIP1-1	Psat3g040640.1	251	7	NPA	NPA	H	I	A	V	6.34	Plas	Vacu
2	PsTIP1-3	Psat3g184440.1	253	6	NPA	NPA	H	I	A	V	5.02	Plas/Vacu	Vacu
3	PsTIP1-4	Psat7g219600.1	253	7	NPA	NPA	H	I	A	V	4.72	Vacu	Vacu
4	PsTIP1-7	Psat6g236600.1	254	6	NPA	NPA	H	I	A	V	5.48	Plas/Vacu	Vacu
5	PsTIP2-1	Psat1g005320.1	250	6	NPA	NPA	H	I	G	R	8.08	Vacu	Vacu
6	PsTIP2-2	Psat4g198360.1	250	6	NPA	NPA	H	I	G	R	5.94	Plas/Vacu	Vacu
7	PsTIP2-3	Psat1g118120.1	256	6	NPA	NPA	H	I	A	L	6.86	Plas/Vacu	Vacu
8	PsTIP2-6	Psat2g177040.1	250	6	NPA	NPA	H	I	G	R	4.93	Vacu	Vacu
9	PsTIP3-2	Psat6g054400.1	260	6	NPA	NPA	H	I	A	R	7.27	Plas/Vacu	Vacu
10	PsTIP4-1	Psat6g037720.2	208	6	NPA	NPA	H	I	A	R	6.29	Vac/ plas	Vacu
11	PsTIP5-1	Psat7g157600.1	254	7	NPA	NPA	N	V	G	C	8.2	Vacu /plas	Vacu/Plas
Nodulin-26 like intrisic proteins (NIPs)
1	PsNIP1-1	Psat1g195040.2	215	5	NPA	0	W	V	F	0	6.71	Plas	Plas
2	PsNIP1-3	Psat1g195800.1	273	5	NPA	NPV	W	V	A	R	6.77	Plas	Plas
3	PsNIP1-5	Psat7g067480.1	276	6	NPA	NPV	W	V	A	N	8.98	Plas	Plas
4	PsNIP1-6	Psat7g067360.1	271	6	NPA	NPA	W	V	A	R	8.65	Plas/Vacu	Plas
5	PsNIP1-7	Psat1g193240.1	277	6	NPA	NPA	W	I	A	R	6.5	Plas/Vacu	Plas
6	PsNIP2-1-2	Psat3g197520.1	115	2	NPA	O	G	0	0	0	9.64	Plas	Plas
7	PsNIP2-2-2	Psat3g197560.1	162	3	0	NPA	0	S	G	R	6.51	Plas	Plas
8	PsNIP3-1	Psat2g072000.1	266	5	NPA	NPA	S	I	A	R	8.59	Plas/Vacu	Plas
9	PsNIP4-1	Psat7g126440.1	276	6	NPA	NPA	W	V	A	R	6.67	Plas	Plas
10	PsNIP4-2	Psat5g230920.1	275	6	NPA	NPA	W	L	A	R	7.01	Plas	Plas
11	PsNIP5-1	Psat6g190560.1	289	5	NPS	NPV	A	I	G	R	7.1	Plas	Plas
12	PsNIP6-1	Psat5g304760.4	162	2	NPA	0	I	0	0	0	9.03	Plas	Plas
13	PsNIP6-2	Psat7g036680.1	254	0	0	0	G	0	0	0	5.27	Chlo	Plas/Nucl
14	PsNIP6-3	Psat7g259640.1	306	6	NPA	NPV	T	I	G	R	8.32	Plas	Plas
15	PsNIP7-1	Psat6g134160.2	503	0	NLK	0	W	G	Q	R	8.5	Vacu	Chlo/Nucl
Small basic intrinsic proteins (SIPs)
1	PsSIP1-1	Psat3g091120.1	246	6	NPT	NPA	V	L	P	N	9.54	Plas	Plas/Vacu
2	PsSIP1-2	Psat1g096840.1	248	5	NTP	NPA	I	V	P	L	9.24	Vacu	Plas/Vacu
3	PsSIP1-3	Psat7g203280.1	240	6	NPS	NPA	N	L	P	N	10.32	Chlo	Plas
4	PsSIP2-1-2	Psat3g082760.1	240	4	NPL	NPA	Y	L	G	S	10.28	Plas	Plas
Uncharacterized X intrinsic proteins (XIPs)
1	PsXIP2-1	Psat7g178080.1	314	6	SPV	NPA	V	V	R	M	7.89	Plas	Plas
Length: protein length (aa); pI: Isoelectric point; Trans-membrane helicase (TMH) represents for the numbers of Trans-membrane helices predicted by TMHMM Server v.2.0 tool; WoLF PSORT and Plant-mPLoc: best possible cellualr localization predicted by the WoLF PSORT and Plant-mPLoc tool, respectively (Chlo Chloroplast, Plas Plasma membrane, Vacu Vacuolar membrane, Nucl Nucleus); LB: Loop B, L: Loop E; NPA: Asparagine-Proline-Alanine; H2 represents for Helix 2, H5 represents for Helix 5, LE1 represents for Loop E1, LE2 represents for Loop E2, Ar/R represents for Aromatic/Arginine.

| Show Table

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Genome distribution and gene structure analysis of pea AQPs

To understand the genome distribution of the 41 PsAQPs, we mapped these genes onto the seven chromosomes of a pea to retrieve their physical locations (Fig. 2). The greatest number (10) of AQPs were found on chromosome 7, whereas the least (2) on chromosome 4 (Fig. 2 and Table 1). Chromosomes 1 and 6 each contain six aquaporin genes, whereas chromosomes 2, 3, and 5 carry four, seven, and four aquaporin genes, respectively (Fig. 2). The trend of clustered distribution of AQPs was seen on specific chromosomes, particularly near the end of chromosome 7.

Figure 2. Chromosomal localization of the 41 PsAQPs on the seven chromosomes of pea. Chr1-7 represents the chromosomes 1 to 7. The numbers on the right of each chromosome show the physical map positions of the AQP genes (Mbp). Blue, green, orange, brown, and black colors represent TIPs, NIPs, PIPs, SIPs, and XIP, respectively.

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The 39 full-length PsAQP proteins have a length of amino acid ranging from 115 to 503 (Table 1) and Isoelectric point (pI) values ranging from 4.72 to 10.35 (Table 2). As a structural signature, transmembrane domains were predicted to exist in all PsAQPs, with the number in individual AQPs varying from 2 to 6. By subfamilies, TIPs harbor the greatest number of TM domains in total, followed by PIPs, NIPs, SIPs, and XIP (Table 2). Exon-intron structure analysis showed that most PsAQPs (16/39) having two introns, while ten members had three, seven members had four, and five members had only one intron (Fig. 3). Overall, PsAQPs exhibited a complex structure with varying intron numbers, positions, and lengths.

Figure 3. The exon-intron structures of the AQP genes in pea. Upstream/downstream region, exon, and intron are represented by a blue box, yellow box, and grey line, respectively.

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Characterization of the NPA motifs

As aforementioned, generally highly conserved two NPA motifs generate an electrostatic repulsion of protons in AQPs to form the water channel, which is essential for the transport of substrate molecules^[15]. In order to comprehend the potential physiological function and substrate specificity of pea aquaporins, NPA motifs (LB, LE) and residues at the ar/R selectivity filter (H2, H5, LE1, and LE2) were examined. (Table 2). We found that all PsTIPs and most PsPIPs had two conserved NPA motifs except for PsPIP1-1, PsPIP2-2-1, and PsPIP2-3, each having a single NPA motif. Among PsNIPs, PsNIP1-6, PsNIP1-6, PsNIP1-7, PsNIP3-1, PsNIP4-1 and PSNIP4-2 had two NPA domains, while PsNIP1-1, PsNIP2-1-2, PsNIP2-2-2 and PsNIP6-1 each had a single NPA motif. In the PsNIP sub-family, the first NPA motif showed an Alanine (A) to Valine (V) substitution in three PsNIPs (PsNIP1-3, PsNIP1-5, and PsNIP6-3) (Table 2). Furthermore, the NPA domains of all members of the XIP and SIP subfamilies were different. The second NPA motif was conserved in PsSIP aquaporins, however, all of the first NPA motifs had Alanine (A) replaced by Leucine (L) (PsSIP2-1-1, PsSIP2-1-2) or Threonine (T) (PsSIP1-1). In comparison to other subfamilies, this motif variation distinguishes water and solute-transporting aquaporins^[45].

Compared to NPA motifs, the ar/R positions were more variable and the amino acid composition appeared to be subfamily-dependent. The majority of PsPIPs had phenylalanine at H2, histidine at H5, threonine at LE1, and arginine at LE2 selective filter (Table 2). All of the PsTIP1 members had a Histidine-Isoleucine-Alanine-Valine structure at this position, while all PsTIP2 members but PsTIP2-3 harbored Histidine-Isoleucine-Glycine-Arginine. Similarly, PsNIPs, PsSIPs and PsXIP also showed subgroup-specific variation in ar/R selectivity filter (Table 2). Each of these substitutions partly determines the function of transporting water^[46].

Predicted subcellular localization of PsAQPs

Sequence-based subcellular localization analysis using WoLF PSORT predicted that all PsPIPs localized in the plasma membrane, which is consistent with their subfamily classification (Table 2). Around half (5/11) of the PsTIPs (PsTIP1-4, PsTIP2-1, PsTIP2-6, PsTIP4-1, and PsTIP5-1) were predicted to localize within vacuoles. However, several TIP members (PsTIP1-1, PsTIP1-3, PsTIP1-7, PsTIP2-2, PsTIP2-3 and PsTIP3-2) were predicted to localize in plasma membranes. We then further investigated their localizations by using another software (Plant-mPLoc, Table 2), which predicted that all the PsTIPs localize within vacuoles, thus supporting that they are tonoplast related. An overwhelming majority of PsNIPs (14/15) and PsXIP were predicted to be found only in plasma membranes., which was also expected (Table 2). Collectively, the versatility in subcellular localization of the pea AQPs is implicative of their distinct roles in controlling water and/or solute transport in the context of plant cell compartmentation.

Tissue expression profiles of PsAQPs

Tissue expression patterns of genes are indicative of their functions. Since there were rich resources of RNA-Seq data from various types of pea tissues in the public database, they were used for the extraction of expression information of PsAQP genes as represented by FPKM values. A heat map was generated to show the expression patterns of PsAQP genes in 18 different tissues/stages and their responses to nitrate levels (Fig. 4). According to the heat map, PsPIP1-2, PsPIP2-3 were highly expressed in root and nodule G (Low-nitrate), whereas PsTIP1-4, PsTIP2-6, and PsNIP1-7 were only expressed in roots in comparison to other tissues. The result also demonstrated that PsPIP1-1 and PsNIP3-1 expressed more abundantly in leaf, tendril, and peduncle, whereas PsPIP2-2-2 and PsTIP1-1 showed high to moderate expressions in all the samples except for a few. Interestingly, PsTIP1-1 expression in many green tissues seemed to be oppressed by low-nitrate. In contrast, some AQPs such as PsTIP1-3, PsTIP1-7, PsTIP5-1, PsNIP1-5, PsNIP4-1, PsNIP5-1, and PsSIP2-1-1 showed higher expression only in the flower tissue. There were interesting developmental stage-dependent regulations of some AQPs in seeds (Fig. 4). For example, PsPIP2-1, PsPIP2-2-1, PsNIP1-6, PsSIP1-1, and PsSIP1-2 were more abundantly expressed in the Seed_12 dap (days after pollination;) tissue than in the Seed_5 dai (days after imbibition) tissue; reversely, PsPIP2-2-2, PsPIP2-4, PsTIP2-3, and PsTIP3-2 showed higher expression in seed_5 dai in compare to seed_12 dap tissues (Fig. 4). The AQP genes may have particular functional roles in the growth and development of the pea based on their tissue-specific expression.

Figure 4. Heatmap analysis of the expression of pea AQP gene expressions in different tissues using RNA-seq data (PRJNA267198). Normalized expression of aquaporins in terms of reads per kilobase of transcript per million mapped reads (RPKM) showing higher levels of PIPs, NIPs, TIPs SIPs, and XIP expression across the different tissues analyzed. (Stage A represents 7-8 nodes; stage B represents the start of flowering; stage D represents germination, 5 d after imbibition; stage E represents 12 d after pollination; stage F represents 8 d after sowing; stage G represents 18 d after sowing, LN: Low-nitrate; HN: High-nitrate.

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PsAQPs expressions in response to osmotic stress and fullerol treatment in imbibing embryos

Expressions of plant AQPs in vegetative tissues under normal and stressed conditions have been extensively studied^[15]; however, little is known about the transcriptional regulation of AQP genes in seeds/embryos. To provide insights into this specific area, wet-bench RNA-Seq was performed on the germinating embryo samples isolated from water (W)-imbibed seeds and those treated with mannitol (M, an osmotic reagent), mannitol, and mannitol plus fullerol (F, a nano-antioxidant). The phenotypic evaluation showed that M treatment had a substantial inhibitory effect on radicle growth, whereas the supplement of F significantly mitigated this inhibition at all concentrations, in particular, 100 mg/mL in MF3, which increased the radicle length by ~33% as compared to that under solely M treatment (Fig. 5). The expression values of PsAQP genes were removed from the RNA-Seq data, and pairwise comparisons were made within the Group 1: W vs M, and Group 2: W vs MF3, where a total of ten and nince AQPs were identified as differentially expressed genes (DEGs), respectively (Fig. 6). In Group 1, six DEGs were up-regulated and four DEGs down-regulated, whereas in Group 2, six DEGs were up-regulated and three DEGs down-regulated. Four genes viz. PsPIPs2-5, PsNIP6-3, PsTIP2-3, and PsTIP3-2 were found to be similarly regulated by M or MF3 treatment (Fig. 6), indicating that their regulation by osmotic stress couldn't be mitigated by fullerol. Three genes, all being PsNIPs (1-1, 2-1-2, and 4-2), were up-regulated only under mannitol treatment without fullerol, suggesting that their perturbations by osmotic stress were migrated by the antioxidant activities. In contrast, four other genes namely PsTIP2-2, PsTIP4-1, PsNIP1-5, and PsSIP1-3 were only regulated under mannitol treatment when fullerol was present.

Figure 5. The visual phenotype and radicle length of pea seeds treated with water (W), 0.3 M mannitol (M), and fullerol of different concentrations dissolved in 0.3 M mannitol (MF). MF1, MF2, MF3, and MF4 indicated fullerol dissolved in 0.3 M mannitol at the concentration of 10, 50, 100, and 500 mg/L, respectively. (a) One hundred and fifty grains of pea seeds each were used for phenotype analysis at 72 h after treatment. Radicle lengths were measured using a ruler in three replicates R1, R2, and R3 in all the treatments. (b) Multiple comparison results determined using the SSR-Test method were shown with lowercase letters to indicate statistical significance (P < 0.05).

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Figure 6. Venn diagram showing the shared and unique differentially expressed PsAQP genes in imbibing seeds under control (W), Mannitol (M) and Mannitol + Fullerol (MF3) treatments. Up-regulation (UG): PsPIP2-5, PsNIP1-1, PsNIP2-1-2, PsNIP4-2, PsNIP6-3, PsNIP1-5, PsTIP2-2, PsTIP4-1, PsSIP1-3, PsXIP2-1; Down-regulation (DG): PsTIP2-3, PsTIP3-2, PsNIP1-7, PsNIP5-1, PsXIP2-1.

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Validation of the DEGs through qRT-PCR

As a validation of the RNA-Seq data, eight genes showing differential expressions in imbibing seeds under M or M + F treatments were selected for qRT-PCR analysis, which was PsTIP4-1, PsTIP2-2, PsTIP2-3, PsTIP3-2, PsPIP2-5, PsXIP2-1, PsNIP6-3 and PsNIP1-5 shown in Fig 6, the expression modes of all the selected genes but PsXIP2-1 were well consistent between the RNA-Seq and the qRT-PCR data. PsXIP2-1, exhibiting slightly decreased expression under M treatment according to RNA-Seq, was found to be up-regulated under the same treatment by qRT-PCR (Fig. 7). This gene was therefore removed from further discussions.

Figure 7. The expression patterns of seven PsAQPs in imbibing seeds as revealed by RNA-Seq and qRT-PCR. The seeds were sampled after 12 h soaking in three different solutions, namely water (W), 0.3 M mannitol (M), and 100 mg/L fullerol dissolved in 0.3 M mannitol (MF3) solution. Error bars are standard errors calculated from three replicates.

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DISCUSSION

This study used the recently available garden pea genome to perform genome-wide identification of AQPs^[35] to help understand their functions in plant growth and development. A total of 39 putative full-length AQPs were found in the garden pea genome, which is very similar to the number of AQPs identified in many other diploid legume crops such as 40 AQPs genes in pigeon pea, chickpea, common bean^[7,47,48], and 44 AQPs in Medicago^[49]. On the other hand, the number of AQP genes in pea is greater compared to diploid species like rice (34)^[4], Arabidopsis thaliana (35)^[3], and 32 and 36 in peanut A and B genomes, respectively^[8]. Phylogenetic analysis assigned the pea AQPs into all five subfamilies known in plants, whereas the presence of only one XIP in this species seems less than the number in other diploid legumes which have two each in common bean and Medicago^[5,48,49]. The functions of the XIP-type AQP will be of particular interest to explore in the future.

The observed exon-intron structures in pea AQPs were found to be conserved and their phylogenetic distribution often correlated with these structures. Similar exon-intron patterns were seen in PIPs and TIPs subfamily of Arabidopsis, soybean, and tomato^[3,6,50]. The two conserved NPA motifs and the four amino acids forming the ar/R SF mostly regulate solute specificity and transport of the substrate across AQPs^[47,51]. According to our analysis, all the members of each AQP subfamilies in garden pea showed mostly conserved NPA motifs and a similar ar/R selective filter. Interestingly, most PsPIPs carry double NPA in LB and LE and a hydrophilic ar/R SF (F/H/T/R) as observed in three legumes i.e., common bean^[48], soybean^[5] chickpea^[7], showing their affinity for water transport. All the TIPs of garden pea have double NPA in LB and LE and wide variation at selectivity filters. Most PsTIP1s (1-1, 1-3, 1-4, and 1-7) were found with H-I-A-V ar/R selectivity filter similar to other species such as Medicago, Arachis, and common bean, that are reported to transport water and other small molecules like boron, hydrogen peroxide, urea, and ammonia^[52]. Compared with related species, the TIPs residues in the ar/R selectivity filter were very similar to those in common bean^[48], Medicago^[49], and Arachis^[8]. In the present study, the NIPs, NIP1s (1-3, 1-5, 1-6, and1-7), and NIP2-2-2 genes have G-S-G-R selectivity. Interestingly, NIP2s with a G-S-G-R selectivity filter plays an important role in silicon influx (Si) in many plant species such as Soybean and Arachis^[6,8]. It was reported that Si accumulation protects plants against various types of biotic and abiotic stresses^[53].

The subcellular localization investigation suggested that most of the PsAQPs were localized to the plasma membrane or vacuolar membrane. The members of the PsPIPs, PsNIPs, and PsXIP subfamilies were mostly located in the plasma membrane, whereas members of the PsTIPs subfamily were often predicted to localize in the vacuolar membrane. Similar situations were reported in many other legumes such as common bean, soybean, and chickpea^[5,7,48]. Apart from that, PsSIPs subfamily were predicted to localize to the plasma membrane or vacuolar membrane, and some AQPs were likely to localize in broader subcellular positions such as the nucleus, cytosol, and chloroplast, which indicates that AQPs may be involved in various molecular transport functions.

AQPs have versatile physiological functions in various plant organs. Analysis of RNA-Seq data showed a moderate to high expression of the PsPIPs in either root or green tissues except for PsPIP2-4, indicating their affinity to water transport. In several other species such as Arachis^[8], common bean^[48], and Medicago^[49], PIPs also were reported to show high expressions and were considered to play an important role to maintain root and leaf hydraulics. Also interestingly, PsTIP2-3 and PsTIP3-2 showed high expressions exclusively in seeds at 5 d after imbibition, indicating their specific roles in seed germination. Earlier, a similar expression pattern for TIP3s was reported in Arabidopsis during the initial phase of seed germination and seed maturation^[54], soybean^[6], canola^[55], and Medicago^[49], suggesting that the main role of TIP3s in regulating seed development is conserved across species.

Carbon nanoparticles such as fullerol have a wide range of potential applications as well as safety concerns in agriculture. Fullerol has been linked to plant protection from oxidative stress by influencing ROS accumulation and activating the antioxidant system in response to drought^[56]. The current study revealed that fullerol at an adequate concentration (100 mg/L), had favorable effects on osmotic stress alleviation. In this study, the radical growth of germinating seeds was repressed by the mannitol treatment, and many similar observations have been found in previous studies^[57]. Furthermore, mannitol induces ROS accumulation in plants, causing oxidative stress^[58]. Our work further validated that the radical growth of germinating seeds were increased during fullerol treatment. Fullerol increased the length of roots and barley seeds, according to Panova et al.^[32]. Fullerol resulted in ROS detoxification in seedlings subjected to water stress^[32].

Through transcriptomic profiling and qRT-PCR, several PsAQPs that responded to osmotic stress by mannitol and a combination of mannitol and fullerol were identified. Most of these differentially expressed AQPs belonged to the TIP and NIP subfamilies. (PsTIP2-2, PsTIP2-3, and PsTIP 3-2) showed higher expression by mannitol treatment, which is consistent with the fact that many TIPs in other species such as GmTIP2;3 and Eucalyptus grandis TIP2 (EgTIP2) also showed elevated expressions under osmotic stress^[54,59]. The maturation of the vacuolar apparatus is known to be aided by the TIPs, which also enable the best possible water absorption throughout the growth of embryos and the germination of seeds^[60]. Here, the higher expression of PsTIP (2-2, 2-3, and 3-2) might help combat water deficiency in imbibing seeds due to osmotic stress. The cellular signals triggering such transcriptional regulation seem to be independent of the antioxidant system because the addition of fullerol didn’t remove such regulation. On the other hand, the mannitol-induced regulation of most PsNIPs were eliminated when fullerol was added, suggesting either a response of these NIPs to the antioxidant signals or being due to the mitigated cellular stress. Based on our experimental data and previous knowledge, we propose that the fullerol-induced up- or down-regulation of specific AQPs belonging to different subfamilies and locating in different subcellular compartments, work coordinatedly with each other, to maintain the water balance and strengthen the tolerance to osmotic stress in germinating pea seeds through reduction of ROS accumulation and enhancement of antioxidant enzyme levels. Uncategorized X intrinsic proteins (XIPs) Aquaporins are multifunctional channels that are accessible to water, metalloids, and ROS.^[32,56]. Due likely to PCR bias, the expression data of PsXIP2-1 from qRT-PCR and RNA-Seq analyses didn’t match well, hampering the drawing of a solid conclusion about this gene. Further studies are required to verify and more deeply dissect the functions of each of these PsAQPs in osmotic stress tolerance.

CONCLUSIONS

A total of 39 full-length AQP genes belonging to five sub-families were identified from the pea genome and characterized for their sequences, phylogenetic relationships, gene structures, subcellular localization, and expression profiles. The number of AQP genes in pea is similar to that in related diploid legume species. The RNA-seq data revealed that PsTIP (2-3, 3-2) showed high expression in seeds for 5 d after imbibition, indicating their possible role during the initial phase of seed germination. Furthermore, gene expression profiles displayed that higher expression of PsTIP (2-3, 3-2) in germinating seeds might help maintain water balance under osmotic stress to confer tolerance. Our results suggests that the biological functions of fullerol in plant cells are exerted partly through the interaction with AQPs.

Deposition of raw data

Under Bio project ID PRJNA793376 at the National Center for Biotechnology Information, raw data of sequencing read has been submitted. The accession numbers for the RNA-seq raw data are stored in GenBank and are mentioned in Supplemental Table S4.

ACKNOWLEDGMENTS

This study is supported by the National Key Research & Development Program of China (2022YFE0198000) and the Key Research Program of Zhejiang Province (2021C02041).

Conflict of interest

Pei Xu is the Editorial Board member of journal Vegetable Research. He was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and his research group.

Rights and permissions
Copyright: © 2022 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.

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Liu Y, Atieno M, Cardoso JA, Yang H, Xu Bin, et al. 2022. Mining and utilization of salinity tolerant legumes in tropical coastal agroecosystems: An overview. Grass Research 2:10 doi: 10.48130/GR-2022-0010

Liu Y, Atieno M, Cardoso JA, Yang H, Xu Bin, et al. 2022. Mining and utilization of salinity tolerant legumes in tropical coastal agroecosystems: An overview. Grass Research 2:10 doi: 10.48130/GR-2022-0010

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Introduction

The rising sea-levels caused by global warming poses a major threat to coastal regions due to salt-water intrusion and damage to agricultural land^[1]. Over the past 20 years, the sea level has risen by about 8 cm^[2]. Based on a new digital elevation model (CoastalDEM) utilizing neural networks, Kulp & Strauss found that the rising sea levels and coastal flooding have tripled as compared to the previous estimate by Shuttle Radar Topography Mission (SRTM)^[3]. About 1 billion people occupy land lying less than 10 m above current high tide lines, most of them living in the Asia and Pacific region, mainly in China, Bangladesh, India, Vietnam, Indonesia, Thailand, Philippines and Japan^[3]. The latest global soil salinity map of 2016 showed that the total area of salt affected land is around 1 billion hectares, an increase of more than 100 Mha from 1986^[4]. Tidal flats occupy an area of about 124,286−131,821 km² globally, with about 11.27% in Indonesia, 9.42% in China, 6.93% in Australia, 4.52% in India and 2.59% in Myanmar^[5].

Increased area of saline soils including large areas of tidal flats have significant impacts on the natural environment and ecosystems as well as socio-economic impacts^[6]. High salinity levels in the soil harm plant growth and limit crop yields. Currently, most of the salt-affected soils, especially coastal saline soils, have been deserted by many farmers leaving extensive areas of idle and unproductive land. This has posed a big challenge to researchers and farmers on how best to fully utilize these saline areas.

Among the various techniques to reclaim saline soil, phytoremediation is generally preferred compared to e.g. hydro or electro reclamation as it is sustainable and economically viable^[7]. Phytoremediation involves the use of plants and associated soil micro-organisms to reduce the harmful effects of contaminants in the environment^[8,9]. For instance, legumes make net nitrogen inputs into ecosystems, and have other multiple environmental benefits by improving soil structure and microbial activity of coastal saline land, and fundamentally improve saline soil^[10]. Salinity tolerant legumes offer the double advantage of reclaiming degraded coastal agroecosystems and providing a local source of livestock feed. The latter is particularly relevant in highly populated countries mainly depending on feed imports, which is often the case in the Asia-Pacific region^[11]. In terms of livestock feed, salinity tolerant legumes improve the quantity and quality of available feed, and have a high nutritional value with high protein content. When grown in areas that are not suitable for crops, they allow farmers to make the most of available land, provide increased income and diversify livelihoods of coastal farmers^[12].

Salinity tolerant legumes grow naturally in many coastal countries, and are receiving increasing attention from researchers and developers due to their adaptation capacities in a range of salinity and drought conditions, and their potential economic benefits^[13,14]. Fully mining and using these plants to improve coastal saline areas and tidal flats is an effective means to overcome impacts of seawater encroachment and increase the livelihoods of 1 billion coastal smallhold farmers^[3].

However, there has been no attempt so far to synthesize knowledge on salinity tolerant legumes and promote their use in coastal ecosystems. Collections and selections have been scattered, mainly in Colombia, China, Australia, India etc.^[15,16]. International Center for Tropical Agriculture (CIAT) (www.tropicalforages.info/text/intro/index.html), Chinese Academy of Tropical Agricultural Sciences (CATAS)^[17], Commonwealth Scientific and Industrial Research Organization (CSIRO) and The International Livestock Research Institute (ILRI) have engaged in the collection and preservation of forage germplasm resources from 1970^[18], but the quantity of salinity tolerant legumes are still low. There is an urgent need to systematically review available coastal forage legume plant resources in order to provide theoretical guidelines for the improvement of coastal saline land. Therefore, the objectives of this study were to (i) review currently available salinity tolerant forage legume species, (ii) discuss their potential for integration into smallholder mixed coastal systems of the Asia-Pacific region, and (iii) present the most promising species in more detail.

Mechanisms of salinity tolerance in legumes

Plant levels

Great breakthroughs have been made in the research on the mechanism of plant salinity tolerance, mainly focusing on model plants Arabidopsis thaliana and crops^[19]. It's clear that salinity tolerance mechanisms of plants are complex traits involving multiple phases and genes^[20]. At the cellular level, salinity tolerant plants maintain this ion balance by excreting Na⁺ out of the cell or compartmentalizing Na⁺ into the vacuole and accumulate osmolytes (such as K⁺, Ca²⁺, proline, soluble sugar, polyamines etc.) in the cytoplasm. Glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane act as Na⁺ receptors for sensing Na⁺ in the apoplastic space and then gate Ca²⁺ influx channels in plants^[21]. With the increase of Ca²⁺, the salt overly sensitive (SOS) signaling pathways (SOS₁, SOS₂, SOS₃) will be activated and play a leading role in mediating the excretion of Na⁺ in plants^[19]. Many other pathways such as MAPK and salinity tolerance related genes (including transcription factors) will also be activated to deal with salinity stress through osmoregulation, ion transport and radical-scavenging. At the tissue or organ level, some salinity tolerant plants usually store Na⁺ in the roots instead of above ground, or old leaves instead of young leaves^[22].

For the salinity tolerant legumes, many of them are belonging to halophytes (plants that survive to complete their life cycle in at least 200 mM salinity)^[23−25], besides the common salinity tolerant mechanisms similar to Arabidopsis thaliana and crops, many of them have special salinity tolerant mechanisms due to their different morphological structure^[26]. Big gaps on the study of salinity tolerant mechanisms based on the whole genome sequencing and lack of studies about the function of special salinity tolerant genes.

Rhizobia level
Rhizobia is a special group of microbes with the unique ability to form symbiotic association with legumes to fix atmospheric nitrogen, a process known as Biological Nitrogen Fixation (BNF)^[27]. Rhizobia-legume association takes place in specialized root structures called nodules, where rhizobia convert atmospheric gaseous dinitrogen (N₂) into ammonia, a form that can be assimilated by the plants; in exchange, the host plant provides carbon compounds to rhizobia^[28−30] . For centuries, this symbiotic association has been used in cropping systems as the most important N-fixing agent^[31,32].

Salt stress can inhibit rhizobia-legume symbiosis by reducing rhizobia growth, nodule formation and BNF due to osmotic stress and high toxic levels of Na⁺ and Cl^- in the cells^[33]. Different rhizobia species exhibit varying levels of tolerance to salt stress. Fast-growing rhizobia species are categorized as more salinity-tolerant e.g. Sinorhizobium meliloti^[34], S. fredii^[35], Mesorhizobium huakuii and Rhizobium tropici^[36] as compared to slow-growing rhizobia such as R. leguminosarum bv. viciae^[37]. Some rhizobia strains have one or several high tolerant abilities to abiotic stress such as high temperature, pH, salinity and drought^[38].

Previous studies have reported rhizobia to be more tolerant to salinity as compared to the host legumes plants, and can enhance tolerance of legumes to salt stress and yield^[39−41]. Salinity-adaptive responses in rhizobia include accumulation of osmoprotectants, enhanced production of exopolysaccharides, expression of stress genes and ROS-scavenging enzymes such as superoxide dismutase^[42,43]. Intracellular accumulation of osmoprotectants such as glycine betaine, glutamate, choline and N-acetyl glutaminyl glutamine amide (NAGGN) have been correlated with salt stress tolerance in Rhizobium meliloti^[44,45]. Salinity tolerance of rhizobia is also related to hormones and protein changes. For example, increased salinity tolerance of Bradyrhizobium strain RJS9-2 may be achieved by production of indole-3-acetic acid (IAA), protein expression and osmoprotectant accumulation^[46]. Salinity tolerant rhizobia may be due to a plasmid-mediated resistance since salinity resistance can be rapidly transferred from tolerant to sensitive rhizobia^[38].

Biofertilizers containing rhizobia are an environmentally friendly approach to enhance soil fertility and legume productivity. Establishment of salinity-tolerant legumes combined with inoculation using salinity-tolerant rhizobia strains is a promising strategy for forage legume production and reclamation of saline soils. Generally, farmers can purchase commercial rhizobia inoculants to apply to the legumes. However, most countries in Asia rely on imported biofertilizers as only few rhizobia inoculants are registered or available in the market, the majority being of poor quality. For instance, by 2018, only 1% of biofertilizers/inoculants registered in China contained rhizobia, mainly produced for Chinese milkvetch (Astragalus sinicus), soybean and peanuts^[33]. In Vietnam, there are very few rhizobia inoculants available in the market^[47], while in Cambodia and Lao PDR, low availability and adoption of rhizobia inoculants have been reported^[33]. This shows a gap and a huge need to develop and promote the use of rhizobia inoculants including salinity-tolerant rhizobia inoculants in this context. Selection of high salinity tolerant legumes and the application of legume-specific, high salinity tolerant rhizobia inoculants has immense potential for successful rhizobium-legume symbiosis and increased N inputs in saline soils.

Diversity of salinity tolerant legumes and integration in smallholder mixed coastal systems

The current known salinity tolerant forage legume species and potentially salinity tolerant species in the tropical regions are summarized in Table 1. Only one species Melilotus indicus (L.) All. is an annual plant, five species are annual or perennial plants, 38 species are perennial plants. Twenty five percent of the 44 species are shrubs, 52% of the 44 species are herbs, 6.8% of the 44 species are trees, the rest have more than one growth habit. No variety was available in the 21 species (47.7% of the total species), indicate that breeding of salinity tolerant legumes needs to be strengthened in the future (Table 1).

Table 1. General information of current salinity tolerant forage legume species.

No.	Scientific name	Common name	Variety	Distribution*	Life cycle	Growth habit
1	Abrus precatorius L.	Rosary pea	―	Tropical zones	Perennial	Shrub
2	Acacia dealbata Link	Mimosa	Dealbata Link, 1846; Mackayana Seem.	World wide	Perennial	Shrub or tree
3	Acacia nilotica (L.) Willd. ex Delile	Gum arabic tree	cupressiformis	Africa	Perennial	Tree
4	Alysicarpus vaginalis (L.) DC.	Alyce Clover, Buffalo clove	Nummularifolius (DC.) Miq., Parvifolius Verdc., stocksii Baker taiwanianus S.S. Ying, Vaginalis, venosa (Blat.& Hall.) A. Pramanik & Thoth.	Tropical zones	Perennial	Herb
5	Arachis pintoi Krapov. & W.C.Greg.	Pinto Peanut	Belomonte, Reyan No.12	Tropical zones of South America	Perennial	Herb
6	Cajanus cajan (L.) Millsp.	Pigeon pea, gungo pea	Phule Tur-12, Babati White, bicolor DC., Cajan, flavus DC.	Tropical zones	Perennial	Shrub
7	Calopogonium mucunoides Desv.	Calopo, wild ground nut	―	Tropical zones	Perennial	Herb
8	Canavalia ensifomis (L.) DC	Jack bean, horse bean	Coriacea Domin, Ensiformis, Normalis Kuntze	Tropical zones	Perennial	Herbaceous vine
9	Canavalia rosea (Sw.) DC.	Bay bean	―	Tropical zones	Perennial	Herbaceous vine
10	Cassia pumila Lam.	―	―	Tropical zones in China, India, Malaysia and Australia	Perennial	Shrub
11	Centrosema pubescens Benth.	Butterfly Pea	Centrosema pubescens Benth. Jinjiang (2019, CATAS)	Tropical zones	Perennial	Herbaceous vine
12	Clitoria ternatea Linn. Sp. Pl.	Asian pigeonwings, blue clitoria, butterfly pea	Angustifolia Hochstetter ex Baker, major Paxton, Pleniflora Fantz, Ternatea	Tropical zones	Perennial	Herbaceous vine
13	Crotalaria albida Heyne ex Roth.	Taiwan crotalaria	albida, Gengmanensis (Z. Wei & C.Y. Yang), Kangrensis A.A. Ansari	Southern Asia	Perennial	Herbaceous
14	Crotalaria bractaeata Roxb. ex DC.	―	―	Southern Asia, America	Perennial	Herb or shrub
15	Crotalaria ferruginea (Grah.) Benth.	Rust-color crotalaria	―	Southern Asia, America	Perennial	Herb
16	Crotalaria retusa Linn.	Large yellow rattlebox, rattleweed	Indica Nampy & Sibichen, retusa, Tunguensis (Lima) Polhill	Tropical zones	Perennial	Shrub
17	Dendrolobium triangulare(Retz.) Schindl.	―	―	Southern Asia	Perennial	Shrub
18	Desmanthus virgatus (L.) Willd.	Wild tantan, Hedge lucerne	Glandulosus (L.) Willd.,1806; Depressus (Humb. & Bonpl. ex Willd.) B.L. Turner; Virgatus	Tropical zones	Perennial	Herb or shrub
19	Erythrina corallodendron L.	Cutlass Bush	Bicolor Krukoff, Connata Krukoff, Corallodendron L,1753	Sporadic spread in tropical zones	Perennial	Tree
20	Galactia elliptifoliola Merr.	―	―	Hainan, China	Perennial	Herbaceous vine
21	Indigofera chuniana Metc.	―	―	Hainan, China	Perennial	Herb or shrub
22	Indigofera enneaphylla Linn. Mant.	―	―	Hainan, China, Indonesia, Papua New Guinea, Australia	Annual or perennial	Herb
23	Indigofera galegoides DC.	―	―	Southern Asia	Perennial	Shrub
24	Indigofera hirsuta Linn.	Hairy Indigo	―	Tropical zones	Annual or perennial	Herb
25	Indigofera litoralis Chun & T.C.Chen	―	―	Tropical zones of China	Perennial	Herb
26	Indigofera suffruticosa Mill.	Anil Indigo	Canescens (J.A. Schmidt) Lobin, Suffruticosa	Tropical zones	Perennial	Shrub
27	Leucaena leucocephala (Lam.) de Wit.	Leucaena, Lead Tree, Cassie	Reyan No. 1	Tropical zones	Perennial	Shrub or tree
28	Macroptilium atropurpureum (L.) Urb.	Purple Bean	Siratro	Tropical zones	Annual or perennial	Herb
29	Melilotus indicus (L.) All.	Annual Melilot, Indian sweet-clover,	Indicus, prostratus P.C. Palau, Tommasini (Jord.) O.E. Schulz	World wide	Annual	Herb
30	Melilotus officinalis (L.) Pall.	Yellow sweet-clover, common yellow melilot	―	World wide	Perennial	Herb
31	Melilotus siculus (Turra) B.D.Jacks.	―	Messina, Neptune	Europe and northern Australia	Annual or perennial	Herb
32	Pongamia pinnata (L.) Pierre	Indian Beech, Pongam tree	Hannii Domin, minor (Benth.) Domin, pinnata, Typica Domin	Southern Asia and northern Australia	Perennial	Tree
33	Pycnospora lutescens (Poir) Schindl.	―	―	Southern Asia and northern Australia	Perennial	Herb or shrub
34	Senna bicapsularis (L.) Roxb.	Christmasbush	Augusti (Harms) H.S. Irwin & Barneby, Bicapsularis	Tropical zones of south America and southern Africa	Perennial	Shrub
35	Sesbania cannabina (Retz.) Pers.	Yellow Pea Bush, Dhaincha	Sericea	Southern Asia and northern Australia	Annual or perennial	Herb or shrub
36	Sesbania rostrata Bremek. & Oberm	―	―	Africa	Annual or perennial	Herb
37	Sesbania sesban (L.) Merr.	Common Sesban	Concolor (Wight & Arn.) Baquar; Bicolor (Wight & Arn.) FW. Andrews; Nubica Chiov; sesban (L.) Merr,1912; Zambesiaca J.B. Gillett	Africa, India, southern America and China	Perennial	Shrub
38	Stylosanthes guianensis (Aubl.) Sw.	Stylo	Reyan No. 20, 21, 22, 24, 25	Tropical zones of Africa, Asia-pacific region and south America	Perennial	Shrub
39	Swainsona formosa	Sturt's Desert Pea	―	Australia	Perennial	Herb
40	Tephrosia purpurea (Linn.) Pers. Syn. Pl.	Sarphonk, wild indigo	Angustissima B.L. Rob., Brevidens Benth., Elongata Craib, Gracilis Tackholm & Boulos, Leptostachya (DC.) Brummitt, Pubescens Baker, Queenslandica Domin, sericea Benth., Yunnanensis Z. Wei	Tropical zones	Perennial	Herb
41	Teramnus labialis (Linn.f.) Spreng.	Blue wiss	Abyssinicus (Hochst. ex A. Rich.) Verdc., Acutus Verdc., Arabicus Verdc., Labialis, Somalensis Vatke	Tropical zones	Perennial	Herb
42	Trifolium fragiferum L.	Strawberry clover	Salina	Subtropical zones of north America, Europe, east Asia, southern Australia and New Zealand	Perennial	Herb
43	Uraria lagopodiodies (Linn.) Desv.ex DC.	―	―	Southern Asia and northern Australia	Perennial	Shrub
44	Vigna marina (Burm.) Merr.	Beachpea	―	tropical zones	Perennial	Herbaceous vine
* Obtained from www.gbif.org/species

There is quite a rich diversity of salinity tolerant species in coastlines of tropical regions, with six different species identified as high salinity tolerant (Table 2, No. 9, 29, 32, 34, 35, 44), 17 as moderate salinity tolerant (Table 2, No. 1−7, 11−12, 16, 30−31, 36−39, 42) and 21 species predicted as salinity tolerant but need to be further proven (Table 2, No. 8, 10, 13−15, 17−28, 33, 40−41, 43).

Table 2. Tolerances of current salinity tolerant forage legume species.

No.	Scientific name	Salinity tolerant	Drought tolerant	Acid tolerant	Shade tolerant	Waterlogging tolerant	Frost tolerant	Reference
1	Abrus precatorius L.	X	X	X	―	―	―	[55]
2	Acacia dealbata Link	X	―	X	―	―	X	[7]
3	Acacia nilotica (L.) Willd. ex Delile	X	X	―	―	―	X	[25]
4	Alysicarpus vaginalis (L.) DC.	X	X	X	―	―	―	https://extension.msstate.edu/content/alyceclover-alysicarpus-vaginalis
5	Arachis pintoi Krapov. & W.C.Greg.	X	X	X	X	―	―	www.tropseeds.com/arachis-pintoi
6	Cajanus cajan (L.) Millsp.	X	X	X	―	―	―	[56]
7	Calopogonium mucunoides Desv.	X	X	―	―	―	―	[57]
8	Canavalia ensifomis (L.) DC	potential*	X	X	―	X	―	pfaf.org/user/Plant.aspx?LatinName=Centrosema+pubescens
9	Canavalia rosea (Sw.) DC.	X X	X	X	―	―	―	[58]
10	Cassia pumila Lam.	Potential*	X	X	―	―	―	[59]
11	Centrosema pubescens Benth.	X	―	―	X	X	―	[60]
12	Clitoria ternatea Linn. Sp. Pl.	X	X	―	X	―	X	[61]
13	Crotalaria albida Heyne ex Roth.	Potential*	X	X	―	―	―	https://baike.baidu.com/item/%E5%93%8D%E9%93%83%E8%B1%86/4860050?fromModule=lemma-qiyi_sense-lemma
14	Crotalaria bractaeata Roxb. ex DC.	Potential*	―	―	―	―	―	―
15	Crotalaria ferruginea (Grah.) Benth.	Potential*	―	―	―	―	―	―
16	Crotalaria retusa Linn.	X	X	―	―	―	―	[62,63]
17	Dendrolobium triangulare(Retz.) Schindl.	Potential*	X	X	―	―	―	[64]
18	Desmanthus virgatus (L.) Willd.	Potential*	―	―	―	―	―	―
19	Erythrina corallodendron L.	Potential*	―	X	X	―	X	https://baike.baidu.com/item/%E9%BE%99%E7%89%99%E8%8A%B1/3567229?fr=kg_general
20	Galactia elliptifoliola Merr.	Potential*	―	―	―	―	―	―
21	Indigofera chuniana Metc.	Potential*	X	―	―	―	―	https://baike.baidu.com/item/%E7%96%8F%E8%8A%B1%E6%9C%A8%E8%93%9D/1483435?fr=kg_general
22	Indigofera enneaphylla Linn. Mant.	Potential*	X	―	―	―	―	https://baike.baidu.com/item/%E4%B9%9D%E5%8F%B6%E6%9C%A8%E8%93%9D/7264010?fr=aladdin
23	Indigofera galegoides DC.	Potential*	―	―	―	―	―	―
24	Indigofera hirsuta Linn.	Potential*	X	―	―	X	―	https://baike.baidu.com/item/%E7%A1%AC%E6%AF%9B%E6%9C%A8%E8%93%9D?fromModule=lemma_search-box
25	Indigofera litoralis Chun & T.C.Chen	Potential*	X	―	―	X	―	https://baike.baidu.com/item/%E6%BB%A8%E6%B5%B7%E6%9C%A8%E8%93%9D/7181556?fr=aladdin
26	Indigofera suffruticosa Mill.	Potential*	―	X	―	―	―	[65]
27	Leucaena leucocephala (Lam.) de Wit.	Potential*	X	―	―	―	―	[25] www.nparks.gov.sg/florafaunaweb/flora/3/4/3471
28	Macroptilium atropurpureum (L.) Urb.	Potential*	X	X	―	X	―	[60]
29	Melilotus indicus (L.) All.	X X	X	―	―	X	―	[66,67]
30	Melilotus officinalis (L.) Pall.	X	―	X	X	―	―	[7,68]
31	Melilotus siculus (Turra) B.D.Jacks.	X	X	―	―	X	―	[7,53,69]
32	Pongamia pinnata (L.) Pierre	X X	X	X	―	X	―	[70]
33	Pycnospora lutescens (Poir) Schindl.	Potential*	―	―	―	―	―	―
34	Senna bicapsularis (L.) Roxb.	X X	X X	―	―	―	―	Data to be published
35	Sesbania cannabina (Retz.) Pers.	X X	X X	―	―	X	―	[14,71,72]
36	Sesbania rostrata Bremek. & Oberm	X	―	X	―	X	―	[7]
37	Sesbania sesban (L.) Merr.	X	―	X	―	―	―	[7,25]
38	Stylosanthes guianensis (Aubl.) Sw.	X	X	X	―	―	―	[73,74]
39	Swainsona formosa	X	X	X	―	―	―	[75]
40	Tephrosia purpurea (Linn.) Pers. Syn. Pl.	Potential*	X	―	―	―	―	https://apps.worldagroforestry.org/treedb/AFTPDFS/Tephrosia_purpurea.PDF
41	Teramnus labialis (Linn.f.) Spreng.	Potential*	―	―	―	―	―	―
42	Trifolium fragiferum L.	X	―	X	―	X	―	[67]
43	Uraria lagopodiodies (Linn.) Desv. ex DC.	Potential*	―	―	―	―	―	―
44	Vigna marina (Burm.) Merr.	X X	X X	X	―	―	―	[13,76]
Potential*: some germplasms collected from coastal areas with potential salinity tolerance and kept in the seed bank of Prataculturae Research Centre, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS). X means stress tolerant; XX means high stress tolerant.

One important additional factor to consider is that some saline soils are marshes, which means that forage legumes integrated in such areas will also need to show waterlogging tolerance. Similar to what was observed for salinity tolerance, many reports have been published to explore legume waterlogging tolerance and legume-rhizobia symbiotic models for waterlogging tolerance^[48−52], legume species Canavalia ensifomis, Centrosema pubescens, Indigofera hirsute, Indigofera litoralis, Macroptilium atropurpureum, Melilotus indicus, Melilotus siculus, Pongamia pinnata, Sesbania cannabina, Sesbania rostrata and Trifolium fragiferum may have the combined tolerance of waterlogging and salinity based on the summarized information (Table 2), previous reports also indicated that Melilotus siculus is a very promising fodder for saline and waterlogged soils^[53,54]. Researchers should pay more attention to legumes combining the traits of salinity and waterlogging tolerance and their field testing.

Some of the listed legume species are tolerant to other abiotic stresses including drought tolerance, acidity tolerance, shade tolerance and frost tolerance (Table 2), drought tolerance is also a very import characteristic compared to salinity and waterlogging.

There is high productivity diversity among the 44 legume species, eight species Alysicarpus vaginalis (4−6 t DW/ha/y), Cajanus cajan (3.175−6.515 t DW/ha/y), Calopogonium mucunoides (4−5 t DW/ha/y), Dendrolobium triangulare (4−5 t DW/ha/y), Indigofera hirsuta (3−6 t DW/ha/y), Macroptilium atropurpureum (2.4−5.5 t DW/ha/y), Stylosanthes guianensis (4−5 t DW/ha/y) and Trifolium fragiferum (4.31−5.19 t DW/ha/y) had relative high productivity compared to other species. The nutritional value of these legumes also varies among species, for instance, the maximum crude protein content of Sesbania sesban, Trifolium fragiferum, Macroptilium atropurpureum and Teramnus labialis are more than 20%. Further evaluation, including other nutrition values, chemical composition and feeding experiments should be conducted to test acceptability to animals and performance of livestock.

Few studies exist regarding the nitrogen fixation efficiency of salinity tolerant legumes even though it is an important indicator for their utilization. Among the 44 species, only 11 species have been reported to have nitrogen fixation efficiency, Sesbania cannabina (949−1,040.25 kg N/ha/y), Arachis pintoi (300 kg/ha/y), Centrosema pubescens (120−270 kg N/ha/y) and Tephrosia purpurea (202.23 kg N/ha/y) had higher nitrogen fixation efficiency^[77−79] (www.tropseeds.com/arachis-pintoi/). We also found very few studies on salinity tolerant rhizobia associated with the listed legumes, most reported rhizobia could tolerate to a salinity concentration higher than 300 mM NaCl (Table 3).

Table 3. Productivity, nutrition value, BNF and salinity tolerance rhizobia of current tolerant forage legume species.

No.	Scientific name	Productivity	Leaf crude protein	Nitrogen fixation efficiency	Salt tolerant rhizobia	Reference
1	Abrus precatorius L.	―	16.28%	―	―	[80]
2	Acacia dealbata Link	1.2−4.0 t DW/ha/y	―	40 kg N/ha/y	―	[81, 82]
3	Acacia nilotica (L.) Willd. ex Delile	3.22 t DW/ha/y	13.92%	―	―	[83−85]
4	Alysicarpus vaginalis (L.) DC.	4–6 t DW/ha/y	17.91%	―	―	https://apps.lucidcentral.org/tropical_forages, https://extension.msstate.edu/content/alyceclover-alysicarpus-vaginalis
5	Arachis pintoi Krapov. & W.C.Greg.	3−4 t DW t/ha/y	17%-20%	300 kg/ha/y	―	www.tropseeds.com/arachis-pintoi/
6	Cajanus cajan (L.) Millsp.	3.175−6.515 t DM/ha/y	―	―	―	[86]
7	Calopogonium mucunoides Desv.	4−5 t DW/ha/y	low	―	―	[57,87]
8	Canavalia ensifomis (L.) DC	―	―	―	―	―
9	Canavalia rosea (Sw.) DC.	1.0−4.6 t DW/ha/y	15%−20%	―	Some strains tolerant to 500−600 mM NaCl	[88,89]
10	Cassia pumila Lam.	―	―	―	―	―
11	Centrosema pubescens Benth.	1.6−2.8 t DW /ha/y	21.36%−23.34%	120−270 kg N/ha/y	―	[78]
12	Clitoria ternatea Linn. Sp. Pl.	1.2−3.6 t DW /ha/y	14%−20%	―	Jd19 Rhizobium strain	[90,91]
13	Crotalaria albida Heyne ex Roth.	―	―	―	―	―
14	Crotalaria bractaeata Roxb. ex DC.	―	―	―	―	―
15	Crotalaria ferruginea (Grah.) Benth.	―	―	―	―	―
16	Crotalaria retusa Linn.	―	14.6%−18.0%	―	―	[63]
17	Dendrolobium triangulare(Retz.) Schindl.	4−5 t DW/ha/y	Average 13.43%	―	―	[92]
18	Desmanthus virgatus (L.) Willd.	3.98 t DW/ha/y	Average 15.20%	―	―	[93]
19	Erythrina corallodendron L.	―	―	―	―	―
20	Galactia elliptifoliola Merr.	2.0−3.5 t DW/ha/y	―	―	―	[94]
21	Indigofera chuniana Metc.	2−4 t DW/ha/y	―	―	―	Date to be published
22	Indigofera enneaphylla Linn. Mant.	3−4 t DW/ha/y	10.7%	―	―	[95]
23	Indigofera galegoides DC.	―	Average 3.26% for total N	―	―	[96]
24	Indigofera hirsuta Linn.	3−6 t DW/ha/y	1.7−1.9% for total N	―	―	[96]
25	Indigofera litoralis Chun & T.C.Chen	―	―	―	―	―
26	Indigofera suffruticosa Mill.	2−4 t DW/ha/y	Average 3.77 for total N	―	―	[96]
27	Leucaena leucocephala (Lam.) de Wit.	3.4 t DW/ha/y	22.8%−25.9%	76 kg N/ha/y	―	[97, 98]
28	Macroptilium atropurpureum (L.) Urb.	2.4–5.5 t DW/ha/y	13.73%−28.2%	62−178 kg N/ha/y	―	[99−101]
29	Melilotus indicus (L.) All.	3.8 t DW/ha/y	―	―	Some strain tolerant to 6% NaCl	[66, 102]
30	Melilotus officinalis (L.) Pall.	―	14.5%−19.4%	Add 80–130 pounds/acre of nitrogen to soil	―	[103, 104] www.sciencedirect.com/topics/agricultural-and-biological-sciences/melilotus-officinalis
31	Melilotus siculus (Turra) B.D.Jacks.	―	―	―	―	―
32	Pongamia pinnata (L.) Pierre	―	―	―	―	―
33	Pycnospora lutescens (Poir) Schindl.	―	―	―	―	―
34	Senna bicapsularis (L.) Roxb.	―	―	―	―	―
35	Sesbania cannabina (Retz.) Pers.	2.46−3.55 t DW/ha/y	―	949−1,040.25 kg N/ha/y	Some strains tolerant to 5.0% (w/v) NaCl	[77, 105, 106] www.healthbenefitstimes.com/sesbania
36	Sesbania rostrata Bremek. & Oberm	1.06−2.19 t DW/ha/y	Average 19.9%	90−219 kg N/ha/y	―	[77,107]
37	Sesbania sesban (L.) Merr.	2.39−2.59 kg DW/ha/y	20%−25%	42.6−109.5 kg N/ha/y	―	[108,109]
38	Stylosanthes guianensis (Aubl.) Sw.	4−5 t DW /ha/y	14%−20%	96−122 kg N/ha/y	Bradyrhizobium strain RJS9-2 tolerant to 350 mM NaCl	[46,110]
39	Swainsona formosa	―	―	―	―	―
40	Tephrosia purpurea (Linn.) Pers. Syn. Pl.	―	16.27%	202.23 kg N/ha/y	Some strains tolerant to 2.5%−3% of NaCl	[79,111]
41	Teramnus labialis (Linn.f.) Spreng.	3−4 t DM/ha/y	22.86%	―	―	[112,113]
42	Trifolium fragiferum L.	4.31−5.19 t DM/ha/y	14.9%−25.7%	―	―	[114,115]
43	Uraria lagopodiodies (Linn.) Desv.ex DC.	―	―	―	―	―
44	Vigna marina (Burm.) Merr.	2.0−4.0 t DW/ha/y	13%−20%	―	Some strains tolerant to 600 mM NaCl	[13,116]

From the 44 summarized legumes, 95.5% have potential to be utilized as forage, Abrus precatorius and Crotalaria retusa contain toxic substances such as monocrotaline which is not safe to use as forage. Eighty four point one percent have potential as green manure, the species Abrus precatorius, Acacia dealbata, Acacia nilotica, Leucaena leucocephala and Pongamia pinnata are too big to be green manure. Eleven point four percent of the species may be developed for food, 59.1% as medicine and 40.9% for ornamental purposes (Table 4).

Table 4. Utilization of current salinity tolerant forage legume species.

No.	Scientific name	Forage	Green manure	Food	Medicine	Ornamental	Reference
1	Abrus precatorius L.	―	―	―	X	X	[118]
2	Acacia dealbata Link	X	―	―	X	X	[119]
3	Acacia nilotica (L.) Willd. ex Delile	X	―	―	X	―	[84]
4	Alysicarpus vaginalis (L.) DC.	X	X	―	X	―	https://apps.lucidcentral.org/tropical_forages
5	Arachis pintoi Krapov. & W.C.Greg.	X	X	―	―	X	https://apps.lucidcentral.org/tropical_forages, www.tropseeds.com/
6	Cajanus cajan (L.) Millsp.	X	X	X	X	―	[120]
7	Calopogonium mucunoides Desv.	X	X	―	―	―	[121]
8	Canavalia ensifomis (L.) DC	X	X	X	X	―	[122]
9	Canavalia rosea (Sw.) DC.	X	X	X	X	X	[89]
10	Cassia pumila Lam.	X	X	―	X	―	[59]
11	Centrosema pubescens Benth.	X	X	―	―	―	[78]
12	Clitoria ternatea Linn. Sp. Pl.	X	X	―	X	X	[90,123]
13	Crotalaria albida Heyne ex Roth.	―	X	―	X	―	[124]
14	Crotalaria bractaeata Roxb. ex DC.	X	X	―	X	―	https://plants.ces.ncsu.edu/plants/crotalaria-spectabilis/
15	Crotalaria ferruginea (Grah.) Benth.	X	X	―	X	―	[125]
16	Crotalaria retusa Linn.	―	X	X	X	―	https://baike.baidu.com/item/%E5%90%8A%E8%A3%99%E8%8D%89?fromModule=lemma_search-box#reference-[1]-3529418-wrap
17	Dendrolobium triangulare(Retz.) Schindl.	X	X	―	X	X	https://baike.baidu.com/item/%E5%81%87%E6%9C%A8%E8%B1%86/23437879?fromModule=lemma-qiyi_sense-lemma
18	Desmanthus virgatus (L.) Willd.	X	X	―	―	―	[93]
19	Erythrina corallodendron L.	X	―	―	X	X	https://baike.baidu.com/item/%E9%BE%99%E7%89%99%E8%8A%B1/3567229?fr=kg_general
20	Galactia elliptifoliola Merr.	X	X	―	―	―	[94]
21	Indigofera chuniana Metc.	X	X	―	―	X	https://baike.baidu.com/item/%E7%96%8F%E8%8A%B1%E6%9C%A8%E8%93%9D/1483435?fr=kg_general
22	Indigofera enneaphylla Linn. Mant.	X	X	―	X	X	[126]
23	Indigofera galegoides DC.	X	X	―	X	X	[96]
24	Indigofera hirsuta Linn.	X	X	―	―	X	[96]
25	Indigofera litoralis Chun & T.C.Chen	X	X	―	X	X	https://baike.baidu.com/item/%E6%BB%A8%E6%B5%B7%E6%9C%A8%E8%93%9D/7181556?fr=aladdin
26	Indigofera suffruticosa Mill.	X	X	―	X	―	[96]
27	Leucaena leucocephala (Lam.) de Wit.	X	―	X	―	X	[97]
28	Macroptilium atropurpureum (L.) Urb.	X	X	―	―	―	[99]
29	Melilotus indicus (L.) All.	X	X	―	X	―	[66]
30	Melilotus officinalis (L.) Pall.	X	X	―	X	X	[103]
31	Melilotus siculus (Turra) B.D.Jacks.	X	X	―	―	X	[10]
32	Pongamia pinnata (L.) Pierre	X	―	―	X	―	[70]
33	Pycnospora lutescens (Poir) Schindl.	X	X	―	―	―	[127]
34	Senna bicapsularis (L.) Roxb.	X	X	―	X	―	[128]
35	Sesbania cannabina (Retz.) Pers.	X	X	―	―	X	[14]
36	Sesbania rostrata Bremek. & Oberm	X	X	―	―	―	[107]
37	Sesbania sesban (L.) Merr.	X	X	―	―	―	[109]
38	Stylosanthes guianensis (Aubl.) Sw.						[110]
39	Swainsona formosa	X	X	―	―	X	[75]
40	Tephrosia purpurea (Linn.) Pers. Syn. Pl.	X	X	―	X	―	https://apps.worldagroforestry.org/treedb/AFTPDFS/Tephrosia_purpurea.PDF
41	Teramnus labialis (Linn.f.) Spreng.	X	X	―	X	―	[112]
42	Trifolium fragiferum L.	X	X	―	―	―	[115]
43	Uraria lagopodiodies (Linn.) Desv.ex DC.	X	X		―	―	https://powo.science.kew.org/taxon/524403-1
44	Vigna marina (Burm.) Merr.	X	X	X	X	X	[129]
X means can be used.

When served as forage, these legumes can either be used in a cut and carry system or grazing, depending on the size of the plots, other crops grown in the same area or farmers' preference. However, in the case of forage, the nutrients contained in the plants do not benefit the soil: there is a trade-off between soil rehabilitation and livelihood benefits^[117]. A compromise is to alternate use for forage or green manure at different times if space allows. A good strategy would be to return animal manure to the soil, eventually following a composting phase. When used as green manure, these legumes can be intercropped with fruit trees or grown in rotation with salinity tolerant food crops (cereals or tubers). They can then protect the soil (moisture conservation, reduce erosion etc.), decompose and increase soil organic matter and nitrogen content.

Adoption of salinity tolerant legumes in coastal areas is limited. Very few studies have reported the adoption of these crops. In Western Australia, salinity tolerant pasture legumes and grasses have been promoted to improve the productivity and profitability of saline land and salt bush-based pastures (www.agric.wa.gov.au/soil-salinity/pasture-legumes-and-grasses-saline-land-western-australia). In south-eastern Tunisia, a study on farmers' willingness to adopt salinity-tolerant forage crops showed that off-farm income availability and flock size significantly affected farmers' willingness to adopt salinity-tolerant forages^[130]. Testing of halophytes and salinity-tolerant plants as potential forage for ruminants was carried in the Near East region, Egypt, but few of them are legumes^[131]. However, adoption barriers typical for cover crops can be expected such as measures related to soil management, impact on yields and income are not immediate, making it difficult for farmers to invest in the technology^[132]. When used as forage, the likelihood of adoption is higher as increases in milk production and weight gain can be quickly observed. One main barrier is the availability of planting material, it's a challenge for the farmers to find planting legumes with high salinity tolerant, high biomass and high nutritive value.

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Mining and utilization of salinity tolerant legumes in tropical coastal agroecosystems: An overview

Abstract

INTRODUCTION

MATERIALS AND METHODS

Identification of AQP genes through Genome-wide

Phylogenetic analysis and classification of AQP genes

Analysis of the NPA motif and transmembrane domains

Characterization of AQP genes and protein properties

In silico tissue expression profiling of AQPs genes

Plant material and seed treatment with different substances

RNA-Seq of imbibing embryos and data analysis

Quantitative real-time PCR (qRT-PCR )

RESULTS

Identification and classification of the AQP genes in garden pea

Genome distribution and gene structure analysis of pea AQPs

Characterization of the NPA motifs

Predicted subcellular localization of PsAQPs

Tissue expression profiles of PsAQPs

PsAQPs expressions in response to osmotic stress and fullerol treatment in imbibing embryos

Validation of the DEGs through qRT-PCR

DISCUSSION

CONCLUSIONS

Deposition of raw data

ACKNOWLEDGMENTS

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