Suppression of OsSAUR2 gene expression immobilizes soil arsenic bioavailability by modulating root exudation and rhizosphere microbial assembly in rice.

One of the factors influencing the behavior of arsenic (As) in environment is microbial-mediated As transformation. However, the detailed regulatory role of gene expression on the changes of root exudation, rhizosphere microorganisms, and soil As occurrence forms remains unclear. In this study, we evidence that loss-of-function of OsSAUR2 gene, a member of the SMALL AUXIN-UP RNA family in rice, results in significantly higher As uptake in roots but greatly lower As accumulation in grains via affecting the expression of OsLsi1, OsLsi2 in roots and OsABCC1 in stems. Further, the alteration of OsSAUR2 expression extensively affects the metabolomic of root exudation, and thereby leading to the variations in the composition of rhizosphere microbial communities in rice. The microbial community in the rhizosphere of Ossaur2 plants strongly immobilizes the occurrence forms of As in soil. Interestingly, Homovanillic acid (HA) and 3-Coumaric acid (CA), two differential metabolites screened from root exudation, can facilitate soil iron reduction, enhance As bioavailability, and stimulate As uptake and accumulation in rice. These findings add our further understanding in the relationship of OsSAUR2 expression with the release of root exudation and rhizosphere microbial assembly under As stress in rice, and provide potential rice genetic resources and root exudation in phytoremediation of As-contaminated paddy soil.

OsLPR5 Encoding Ferroxidase Positively Regulates the Tolerance to Salt Stress in Rice.

Salinity is a major abiotic stress that harms rice growth and productivity. Low phosphate roots (LPRs) play a central role in Pi deficiency-mediated inhibition of primary root growth and have ferroxidase activity. However, the function of LPRs in salt stress response and tolerance in plants remains largely unknown. Here, we reported that the OsLPR5 was induced by NaCl stress and positively regulates the tolerance to salt stress in rice. Under NaCl stress, overexpression of OsLPR5 led to increased ferroxidase activity, more green leaves, higher levels of chlorophyll and lower MDA contents compared with the WT. In addition, OsLPR5 could promote the accumulation of cell osmotic adjustment substances and promote ROS-scavenging enzyme activities. Conversely, the mutant lpr5 had a lower ferroxidase activity and suffered severe damage under salt stress. Moreover, knock out of OsLPR5 caused excessive Na+ levels and Na+/K+ ratios. Taken together, our results exemplify a new molecular link between ferroxidase and salt stress tolerance in rice.

Mutational Bias and Natural Selection Driving the Synonymous Codon Usage of Single-Exon Genes in Rice (Oryza sativa L.).

The relative abundance of single-exon genes (SEGs) in higher plants is perplexing. Uncovering the synonymous codon usage pattern of SEGs will benefit for further understanding their underlying evolutionary mechanism in plants. Using internal correspondence analysis (ICA), we reveal a significant difference in synonymous codon usage between SEGs and multiple-exon genes (MEGs) in rice. But the effect is weak, accounting for only 2.61% of the total codon usage variability. SEGs and MEGs contain remarkably different base compositions, and are under clearly differential selective constraints, with the former having higher GC content, and evolving relatively faster during evolution. In the group of SEGs, the variability in synonymous codon usage among genes is partially due to the variations in GC content, gene function, and gene expression level, which accounts for 22.03%, 5.99%, and 3.32% of the total codon usage variability, respectively. Therefore, mutational bias and natural selection should work on affecting the synonymous codon usage of SEGs in rice. These findings may deepen our knowledge for the mechanisms of origination, differentiation and regulation of SEGs in plants.

The miR528-D3 Module Regulates Plant Height in Rice by Modulating the Gibberellin and Abscisic Acid Metabolisms.

Plant height, as one of the important agronomic traits of rice, is closely related to yield. In recent years, plant height-related genes have been characterized and identified, among which the DWARF3 (D3) gene is one of the target genes of miR528, and regulates rice plant height and tillering mainly by affecting strigolactone (SL) signal transduction. However, it remains unknown whether the miR528 and D3 interaction functions in controlling plant height, and the underlying regulatory mechanism in rice. In this study, we found that the plant height, internode length, and cell length of internodes of d3 mutants and miR528-overexpressing (OE-miR528) lines were greatly shorter than WT, D3-overexpressing (OE-D3), and miR528 target mimicry (OE-MIM528) transgenic plants. Knockout of D3 gene (d3 mutants) or miR528-overexpressing (OE-miR528) triggers a substantial reduction of gibberellin (GA) content, but a significant increase of abscisic acid (ABA) accumulation than in WT. The d3 and OE-miR528 transgenic plants were much more sensitive to GA, but less sensitive to ABA than WT. Moreover, the expression level of GA biosynthesis-related key genes, including OsCPS1, OsCPS2, OsKO2 and OsKAO was remarkably higher in OE-D3 plants, while the NECD2 expression, a key gene involved in ABA biosynthesis, was significantly higher in d3 mutants than in WT and OE-D3 plants. The results indicate that the miR528-D3 module negatively regulates plant height in rice by modulating the GA and ABA homeostasis, thereby further affecting the elongation of internodes, and resulting in lower plant height, which adds a new regulatory role to the D3-mediated plant height controlling in rice.

The miR528-AO Module Confers Enhanced Salt Tolerance in Rice by Modulating the Ascorbic Acid and Abscisic Acid Metabolism and ROS Scavenging.

The monocot lineage-specific miR528 was previously established as a multistress regulator. However, it remains largely unclear how miR528 participates in response to salinity stress in rice. Here, we show that miR528 positively regulates rice salt tolerance by down-regulating a gene encoding l-ascorbate oxidase (AO), thereby bolstering up the AO-mediated abscisic acid (ABA) synthesis and ROS scavenging. Overexpression of miR528 caused a substantial increase in ascorbic acid (AsA) and ABA contents but a significant reduction in ROS accumulation, resulting in the enhanced salt tolerance of rice plants. Conversely, knockdown of miR528 or overexpression of AO stimulated the expression of the AO gene, hence lowering the level of AsA, a critical antioxidant that promotes the ABA content but reduces the ROS level, and then compromising rice tolerance to salinity. Together, the findings reveal a novel mechanism of the miR528-AO module-mediated salt tolerance by modulating the processes of AsA and ABA metabolism as well as ROS detoxification, which adds a new regulatory role to the miR528-AO stress defense pathway in rice.

Evolutionary divergence of function and expression of laccase genes in plants.

Laccases (LACs) are versatile enzymes that catalyze oxidation of a wide range of substrates, thereby functioning in regulation of plant developmental processes and stress responses. However, with a few exceptions, the function of most LACs remains unclear in plants. In this study, we newly identified 4, 12, 22, 26, 27, 28 and 49 LAC genes for Physcomitrella patens, Amborella trichopoda, Zeamays, Ricinus communis, Vitis vinifera, Triticum aestivum and Glycine max, on the basis of exhaustive homologous sequence searches. In these plants, LACs differ greatly in sequence length and physical properties, such as molecular weight and theoretical isoelectric point (pI), but majority of them contain a signal peptide at their N-terminus. The originality of LACs could be traced back to as early as the emergence of moss. Plant LACs are clearly divided into seven distinct classes, where six ancient LACs should be present prior to the divergence of gymnosperms and angiosperms. Functional divergence analysis reveal that functional differentiation should occur among different groups of LACs because of altered selective constraints working on some critical amino acid sites (CAASs) within conserved laccase domains during evolution. Soybean and maize LACs have significantly different exon frequency (6.08 vs 4.82), and they are unevenly distributed and tend to form gene clusters on some chromosomes. Further analysis shows that the expansion of LAC gene family would be due toextensive tandem and chromosomal segmental duplications in the two plant species. Interestingly, *81.6% and 36.4% of soybean and maize LACs are potential targets of miRNAs, such as miR397a/b, miR408d, or miR528a/b etc. Both soybean and maize LACs are tissue specifically and developmental-specifically expressed, and are in response to different external abiotic and biotic stressors. These results suggest a diversity of functions of plant LAC genes, which will broaden our understanding and lay solid foundation for further investigating their biological functions in plants.

The genome-wide dynamics of purging during selfing in maize.

Self-fertilization (also known as selfing) is an important reproductive strategy in plants and a widely applied tool for plant genetics and plant breeding. Selfing can lead to inbreeding depression by uncovering recessive deleterious variants, unless these variants are purged by selection. Here we investigated the dynamics of purging in a set of eleven maize lines that were selfed for six generations. We show that heterozygous, putatively deleterious single nucleotide polymorphisms are preferentially lost from the genome during selfing. Deleterious single nucleotide polymorphisms were lost more rapidly in regions of high recombination, presumably because recombination increases the efficacy of selection by uncoupling linked variants. Overall, heterozygosity decreased more slowly than expected, by an estimated 35% to 40% per generation instead of the expected 50%, perhaps reflecting pervasive associative overdominance. Finally, three lines exhibited marked decreases in genome size due to the purging of transposable elements. Genome loss was more likely to occur for lineages that began with larger genomes with more transposable elements and chromosomal knobs. These three lines purged an average of 398 Mb from their genomes, an amount equivalent to three Arabidopsis thaliana genomes per lineage, in only a few generations.

Demography and its effects on genomic variation in crop domestication.

Over two thousand plant species have been modified morphologically through cultivation and human use. Here, we review three aspects of crop domestication that are currently undergoing marked revisions, due to analytical advancements and their application to whole genome resequencing (WGS) data. We begin by discussing the duration and demographic history of domestication. There has been debate as to whether domestication occurred quickly or slowly. The latter is tentatively supported both by fossil data and application of WGS data to sequentially Markovian coalescent methods that infer the history of effective population size. This history suggests the possibility of extended human impacts on domesticated lineages prior to their purposeful cultivation. We also make the point that demographic history matters, because it shapes patterns and levels of extant genetic diversity. We illustrate this point by discussing the evolutionary processes that contribute to the empirical observation that most crops examined to date have more putatively deleterious alleles than their wild relatives. These deleterious alleles may contribute to genetic load within crops and may be fitting targets for crop improvement. Finally, the same demographic factors are likely to shape the spectrum of structural variants (SVs) within crops. SVs are known to underlie many of the phenotypic changes associated with domestication and crop improvement, but we currently lack sufficient knowledge about the mechanisms that create SVs, their rates of origin, their population frequencies and their phenotypic effects.

Deleterious Variants in Asian Rice and the Potential Cost of Domestication.

Many SNPs are predicted to encode deleterious amino acid variants. These slightly deleterious mutations can provide unique insights into population history, the dynamics of selection, and the genetic bases of phenotypes. This is especially true for domesticated species, where a history of bottlenecks and selection may affect the frequency of deleterious variants and signal a "cost of domestication". Here, we investigated the numbers and frequencies of deleterious variants in Asian rice (Oryza sativa), focusing on two varieties (japonica and indica) and their wild relative (O. rufipogon). We investigated three signals of a potential cost of domestication in Asian rice relative to O. rufipogon: an increase in the frequency of deleterious SNPs (dSNPs), an enrichment of dSNPs compared with synonymous SNPs (sSNPs), and an increased number of deleterious variants. We found evidence for all three signals, and domesticated individuals contained ∼3-4% more deleterious alleles than wild individuals. Deleterious variants were enriched within low recombination regions of the genome and experienced frequency increases similar to sSNPs within regions of putative selective sweeps. A characteristic feature of rice domestication was a shift in mating system from outcrossing to predominantly selfing. Forward simulations suggest that this shift in mating system may have been the dominant factor in shaping both deleterious and neutral diversity in rice.

Involvement of miR528 in the Regulation of Arsenite Tolerance in Rice (Oryza sativa L.).

Tens of miRNAs were previously established as being arsenic (As) stress responsive in rice. However, their functional role in As tolerance remains unclear. This study demonstrates that transgenic plants overexpressing miR528 (Ubi::MIR528) were more sensitive to arsenite [As(III)] compared with wild-type (WT) rice. Under normal and stress conditions, miR528-5p and -3p were highly up-regulated in both the roots and leaves of transgenic plants, which exhibited a negative correlation with the expression of seven target genes. Compared with WT plants, Ubi::MIR528 plants showed excessive oxidative stress generation and remarkable amino acid content changes in the roots and leaves upon As(III) exposure. Notably, the expression profiles of diverse functional genes were clearly different between WT and transgenic plants. Thus, the observed As(III) sensitivity of Ubi::MIR528 plants was likely due to the strong alteration of antioxidant enzyme activity and amino acid profiles and the impairment of the As(III) uptake, translocation, and tolerance systems of rice.

Genome-wide identification and evolutionary analysis of positively selected miRNA genes in domesticated rice.

The next-generation sequencing of tens to hundreds of plant genotypes made the uncovering of miRNA genes evolution available at the genome-wide level. Using the combinations of population genetics and evolutionary biology approaches, we have identified 21 miRNA loci having significant negative Tajima's D and Fu and Li's D* and F* values, of which 14 miRNAs (ps-miRNAs) showing clear signatures of positive selection in domesticated rice. The average sequence diversity (π) of the 21 miRNAs in cultivated rice is only 13.8 % of that in their wild progenitors. Interestingly, protein-coding genes immediately flanking these ps-miRNAs are apparently under weaker selective constraints. Totally, the 21 miRNAs are predicted to target 68 mRNA genes, of which 12 targets are estimated to have endured positive selection during rice evolution. In addition, the expression pattern and potential biological functions of ps-miRNAs targets are further investigated by searching published micro-array data and different mutant databases, respectively. We conclude that miRNAs, like protein-coding genes, should be crucial for driving rice evolution. These analyses may deepen our understanding on the miRNA genes evolution and functions during rice domestication.

Mutational bias is the driving force for shaping the synonymous codon usage pattern of alternatively spliced genes in rice (Oryza sativa L.).

Alternative splicing plays important roles in diverse aspects of plant development, metabolism, and stress responses. However, the regulatory mechanisms of alternative splicing of genes still remain incompletely elucidated, especially in plants. In this study, the synonymous codon usage pattern of alternatively spliced (AS) genes in rice was firstly explored using the combination of correspondence analysis (CA), internal CA, correlation and ANOVA analyses. The results show that alternatively and non-alternatively spliced (non-AS) genes have similar tendency for overall codon usage, but exhibit significant difference in 58 out of 64 codons. AS and non-AS genes are both under strong purifying selection, but the former ones have significant lower mutation rate and are prone to be enriched towards the chromosomal ends. In the group of AS genes, the variability in synonymous codon usage between genes is mainly due to the variations in GC content, CDS length, as well as gene functions. Mutational bias that accounts for 25.85 % of the total codon usage variability plays a major role in shaping the codon usage pattern of AS genes. In contrast, no obvious evidence is found for the contributions of translational selection, AS types, the conservation of AS events, and numbers of AS variants to the codon usage divergence between AS genes. These findings may be useful for further understanding the mechanisms of origination, differentiation and regulation of alternatively spliced genes in plants.

Genome-wide identification and analysis of miRNA-related single nucleotide polymorphisms (SNPs) in rice.

BACKGROUND: MiRNAs are key regulators in the miRNA-mediated regulatory networks. Single nucleotide polymorphisms (SNPs) that occur at miRNA-related regions may cause serious phenotype changes. To gain new insights into the evolution of miRNAs after SNP variation, we performed a genome-wide scan of miRNA-related SNPs, and analyzed their effects on the stability of miRNAs structure and the alteration of target spectrum in rice. RESULTS: We find that the SNP density in pre-miRNAs is significantly higher than that in the flanking regions, owing to the rapid evolution of a large number of species-specific miRNAs in rice. In contrast, it is obvious that deeply conserved miRNAs are under strong purifying selection during evolution. In most cases, the SNPs in stem regions may result in the miRNA hairpin structures changing from stable to unstable status; And SNPs in mature miRNAs have great potential to have either newly created or disrupted the miRNA-target interactions. However, the total number of gained targets is over 2.5 times greater than that are lost after mutation. Notably, 12 putative domestication-related miRNAs have been identified, where the SNP density is significantly lower. CONCLUSIONS: The present study provides the first outline of SNP variations occurred in rice pre-miRNAs at the whole genome-wide level. These analyses may deepen our understanding on the effects of SNPs on the evolution of miRNAs in the rice genome.

Effect of silicate supplementation on the alleviation of arsenite toxicity in 93-11 (Oryza sativa L. indica).

Chronic exposure to arsenic (As) in rice has raised many health and environmental problems. As reported, great variation exists among different rice genotypes in As uptake, translocation, and accumulation. Under hydroponic culture, we find that the Chinese wild rice (Oryza rufipogon; acc. 104624) takes up the most arsenic among tested genotypes. Of the cultivated rice, the indica cv. 93-11 has the lowest arsenic translocation factor value but accumulates the maximum concentration of arsenic followed by Nipponbare, Minghui 86, and Zhonghua 11. Higher level of arsenite concentration (50 µM) can induce extensive photosynthesis and root growth inhibition, and cause severe oxidative stress. Interestingly, external silicate (Si) supplementation has significantly increased the net photosynthetic rate, and promoted root elongation, as well as strongly ameliorated the oxidative stress by increasing the activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and peroxidase in roots and/or leaves of 93-11 seedlings. Notably, 1.873 mM concentration of Si considerably decreases the total As uptake and As content in roots, but significantly increases the As translocation from roots to shoots. In contrast, Si supplementation with 1.0 mM concentration significantly increases the total As uptake and As concentrations in roots and shoots of 93-11 seedlings after 50 µM arsenite treatment for 6 days.

De novo characterization of the Chinese fir (Cunninghamia lanceolata) transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.

BACKGROUND: Chinese fir (Cunninghamia lanceolata) is an important timber species that accounts for 20-30% of the total commercial timber production in China. However, the available genomic information of Chinese fir is limited, and this severely encumbers functional genomic analysis and molecular breeding in Chinese fir. Recently, major advances in transcriptome sequencing have provided fast and cost-effective approaches to generate large expression datasets that have proven to be powerful tools to profile the transcriptomes of non-model organisms with undetermined genomes. RESULTS: In this study, the transcriptomes of nine tissues from Chinese fir were analyzed using the Illumina HiSeq™ 2000 sequencing platform. Approximately 40 million paired-end reads were obtained, generating 3.62 gigabase pairs of sequencing data. These reads were assembled into 83,248 unique sequences (i.e. Unigenes) with an average length of 449 bp, amounting to 37.40 Mb. A total of 73,779 Unigenes were supported by more than 5 reads, 42,663 (57.83%) had homologs in the NCBI non-redundant and Swiss-Prot protein databases, corresponding to 27,224 unique protein entries. Of these Unigenes, 16,750 were assigned to Gene Ontology classes, and 14,877 were clustered into orthologous groups. A total of 21,689 (29.40%) were mapped to 119 pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The majority of the genes encoding the enzymes in the biosynthetic pathways of cellulose and lignin were identified in the Unigene dataset by targeted searches of their annotations. And a number of candidate Chinese fir genes in the two metabolic pathways were discovered firstly. Eighteen genes related to cellulose and lignin biosynthesis were cloned for experimental validating of transcriptome data. Overall 49 Unigenes, covering different regions of these selected genes, were found by alignment. Their expression patterns in different tissues were analyzed by qRT-PCR to explore their putative functions. CONCLUSIONS: A substantial fraction of transcript sequences was obtained from the deep sequencing of Chinese fir. The assembled Unigene dataset was used to discover candidate genes of cellulose and lignin biosynthesis. This transcriptome dataset will provide a comprehensive sequence resource for molecular genetics research of C. lanceolata.

Mutational bias and translational selection shaping the codon usage pattern of tissue-specific genes in rice.

The regulatory mechanisms of determining which genes specifically expressed in which tissues are still not fully elucidated, especially in plants. Using internal correspondence analysis, I first establish that tissue-specific genes exhibit significantly different synonymous codon usage in rice, although this effect is weak. The variability of synonymous codon usage between tissues accounts for 5.62% of the total codon usage variability, which has mainly arisen from the neutral evolutionary forces, such as GC content variation among tissues. Moreover, tissue-specific genes are under differential selective constraints, inferring that natural selection also contributes to the codon usage divergence between tissues. These findings may add further evidence in understanding the differentiation and regulation of tissue-specific gene products in plants.

Molecular identification and analysis of arsenite stress-responsive miRNAs in rice.

Arsenic is highly toxic to living organisms including humans and plants. To investigate the responsive functions of miRNAs under arsenite stress, an indica rice, Minghui 86, has been deeply sequenced, and a total of 67 arsenite-responsive miRNAs were identified in rice seedling roots, of which 5 were further validated experimentally. The potential targets of those differential miRNAs include some transcription factors, protein kinases, and DNA- or metal ion-binding proteins that are associated with cellular and metabolic processes, pigmentation, and stress responses. The regulatory roles of four miRNAs on their targets in response to arsenite were further confirmed by real time RT-PCR. Interestingly, osa-miR6256 was originally characterized as a putative exonic miRNA, supporting the notion that miRNAs may also originate from some exons in plants. The first identification of arsenite-responsive miRNAs at the whole genome-wide level will broaden the current understanding of the molecular mechanisms of arsenite responses in rice.

Analyses of the sucrose synthase gene family in cotton: structure, phylogeny and expression patterns.

BACKGROUND: In plants, sucrose synthase (Sus) is widely considered as a key enzyme involved in sucrose metabolism. Several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, while limited information of Sus genes is available to date for cotton. RESULTS: Here, we report the molecular cloning, structural organization, phylogenetic evolution and expression profiles of seven Sus genes (GaSus1 to 7) identified from diploid fiber cotton (Gossypium arboreum). Comparisons between cDNA and genomic sequences revealed that the cotton GaSus genes were interrupted by multiple introns. Comparative screening of introns in homologous genes demonstrated that the number and position of Sus introns are highly conserved among Sus genes in cotton and other more distantly related plant species. Phylogenetic analysis showed that GaSus1, GaSus2, GaSus3, GaSus4 and GaSus5 could be clustered together into a dicot Sus group, while GaSus6 and GaSus7 were separated evenly into other two groups, with members from both dicot and monocot species. Expression profiles analyses of the seven Sus genes indicated that except GaSus2, of which the transcripts was undetectable in all tissues examined, and GaSus7, which was only expressed in stem and petal, the other five paralogues were differentially expressed in a wide ranges of tissues, and showed development-dependent expression profiles in cotton fiber cells. CONCLUSIONS: This is a comprehensive study of the Sus gene family in cotton plant. The results presented in this work provide new insights into the evolutionary conservation and sub-functional divergence of the cotton Sus gene family in response to cotton fiber growth and development.

Novel miRNAs in the control of arsenite levels in rice.

In a species, the miRNA repertoire comprises many lineage- or species-specific miRNAs. Using deep sequencing, the whole transcriptome of seedling roots was studied and a total of 18 novel miRNAs in indica rice Minghui 86 were identified. Among these novel miRNAs six were up-regulated and two were down-regulated under arsenite stress. Quantitative real-time RT-PCR analysis revealed that miR6254 was predominantly expressed in roots relative to other tissues, whereas miR6250 and miR169i-3p were constitutively expressed in all tissues examined, with high abundance in roots (miR6250) or leaves (miR169i-3p). The miR6250 and miR169i-3p miRNAs also exhibited distinct expression patterns in rice cultivars Minghui 86 and Nipponbare at different time points after arsenite treatment. The predicted targets for these miRNAs included some protein kinases, DNA, or ATP-binding proteins. Besides arsenite, the expression of targets of miR6250 and miR6254 was also up- or down-regulated in response to abiotic environmental stresses, indicative of their involvement in regulation of plant adaptation. Three types of cis-elements involved in hormone, light, and stress response were found to occur frequently in the promoter regions. Interestingly, miR6254 was originally characterized to be an exonic miRNA located in the exon of AK101391, supporting the notion that miRNAs may also originate from some exons in plants.

Functional divergence of the NIP III subgroup proteins involved altered selective constraints and positive selection.

BACKGROUND: Nod26-like intrinsic proteins (NIPs) that belong to the aquaporin superfamily are unique to plants. According to homology modeling and phylogenetic analysis, the NIP subfamily can be further divided into three subgroups with distinct biological functions (NIP I, NIP II, and NIP III). In some grasses, the NIP III subgroup proteins (NIP2s) were demonstrated to be permeable to solutes with larger diameter, such as silicic acid and arsenous acids. However, to date there is no data-mining or direct experimental evidences for the permeability of such larger solutes for dicot NIP2s, although they exhibit similar three-dimensional structures as those in grasses. It is therefore intriguing to investigate the molecular mechanisms that drive the evolution of plant NIP2s. RESULTS: The NIP III subgroup is more ancient with a divergence time that predates the monocot-dicot split. The proliferation of NIP2 genes in modern grass species is primarily attributed to whole genome and segmental chromosomal duplication events. The structure of NIP2 genes is relatively conserved, possessing five exons and four introns. All NIP2s possess an ar/R filter consisting of G, S, G, and R, except for the cucumber CsNIP2;2, where a small G in the H2 is substituted with the bulkier C residue. Our maximum likelihood analysis revealed that NIP2s, especially the loop A (LA) region, have undergone strong selective pressure for adaptive evolution. The analysis at the amino acid level provided strong statistical evidences for the functional divergence between monocot and dicot NIP III subgroup proteins. In addition, several SDPs (Specificity Determining Positions) responsible for functional specificity were predicted. CONCLUSIONS: The present study provides the first evidences of functional divergence between dicot and monocot NIP2s, and suggests that positive selection, as well as a radical shift of evolutionary rate at some critical amino acid sites is the primary driver. These findings will expand our understanding to evolutionary mechanisms driving the functional diversification of monocot and dicot NIP III subgroup proteins.