Pan-plantae aquaporin evolution: insights into PIP2 as a potential regulator of rose prickle development

Abstract

Plant aquaporin (AQP), one of the ancient protein superfamilies with high diversity, plays a canonical role as water channel transporters to regulate seed germination, stomatal movement, cell elongation, reproduction, and environmental adaptation. Previously, we have demonstrated that two AQPs colocalize within a quantitative trait locus (QTL) linked to the genetic regulation of rose prickles, which function as water reservoirs at their early developmental stages. Here, we systematically characterize the diversity of AQPs in all plant lineages taking the Rosaceae as one example and establish a genetic link between PIP2;1 expression and prickle development. We reveal the ancient origin and remarkable deep conservation in AQP protein structure, the increase of copy number following the diversification of plant lineages and a strong positive selection of the PIP2 and the SIP subfamilies within Rosaceae. Intriguingly, this is contrasted by substantial expression divergence, which is accompanied with regulatory divergence, particularly in cis-regulatory elements associated with hormone responses and developmental regulation. Spatial–temporal expression profiling demonstrates dynamic AQP expression patterns during development of prickle and the underlying cortical tissue in the prickly Rosa gigantea, while also differ substantially in shoots and leaves of the prickly Rosa chinensis and prickle-free Rosa wichuraiana. Silencing the expression of PIP2;1 within the prickle QTL region significantly attenuates the prickle production along rose stems. Our findings not only highlight both evolutionary conservation in AQP protein architecture and lineage-specific regulatory innovation in Rosaceae but also uncover a novel regulatory role of PIP2;1 in epidermal differentiation of the non-model roses, and of likely many other spiny angiosperms.

References

[1] Maurel C, Verdoucq L, Rodrigues O . Aquaporins and plant transpiration. Plant Cell Environ. 2016; 39: 2580-7

[2] Gao L, Lu Z, Ding L. et al. Role of aquaporins in determining carbon and nitrogen status in higher plants. Int J Mol Sci. 2018; 19: 35

[3] Ariani A, Francini A, Andreucci A. et al. Over-expression of AQUA1 in Populus alba Villafranca clone increases relative growth rate and water use efficiency, under Zn excess condition. Plant Cell Rep. 2016; 35: 289-301

[4] Patankar HV, Al-Harrasi I, Al-Yahyai R. et al. Functional characterization of date palm aquaporin gene PdPIP1;2 confers drought and salinity tolerance to yeast and Arabidopsis. Genes (Basel). 2019; 10: 390

[5] Quiroga G, Erice G, Aroca R. et al. Enhanced drought stress tolerance by the arbuscular mycorrhizal symbiosis in a drought-sensitive maize cultivar is related to a broader and differential regulation of host plant aquaporins than in a drought-tolerant cultivar. Front Plant Sci. 2017; 8: 1056

[6] Duric MJ, Subotić AR, Prokić LT. et al. Molecular characterization and expression of four aquaporin genes in Impatiens walleriana during drought stress and recovery. Plants (Basel). 2021; 10: 154

[7] Abdelhakam S, Rabei S, Nada R. et al. Role of aquaporins in salt tolerance of common sunflower. Sci J Damietta Fac Sci. 9: 20-31

[8] Chen X, Jin S, du H. et al. Plasma membrane CYBDOM proteins catalyse apoplastic AsA regeneration and interact with RbohD to activate autophagy and drought tolerance in plants. Nat Plants. 2025; 11: 1681-99

[9] Duan H, Cui G, Li Y. et al. A T2T gap-free genome provides insights into tetraploid Salicornia europaea evolution and aquaporin function in stress tolerance. Plant Physiol. 2025; 199: kiaf565

[10] Xu C, Wang M, Zhou L. et al. Heterologous expression of the wheat aquaporin gene TaTIP2;2 compromises the abiotic stress tolerance of Arabidopsis thaliana. PLoS One. 2013; 8: e79618

[11] Ayadi M, Brini F, Masmoudi K . Overexpression of a wheat aquaporin gene, TdPIP2;1, enhances salt and drought tolerance in transgenic durum wheat cv. Maali. Int J Mol Sci. 2019; 20: 2389

[12] Ruiz-Lozano JM, Quiroga G, Erice G. et al. Using the maize nested association mapping (NAM) population to partition arbuscular mycorrhizal effects on drought stress tolerance into hormonal and hydraulic components. Int J Mol Sci. 2022; 23: 9822

[13] Berna-Sicilia JA, Quizhpe-Romero M, Hurtado-Navarro M. et al. Combined soil microorganism amendments and foliar micronutrient nanofertilization increased the production of Allium cepa L. through aquaporin gene regulation. Life (Basel). 2023; 14: 4

[14] Wang M, Ding L, Gao L. et al. The interactions of aquaporins and mineral nutrients in higher plants. Int J Mol Sci. 2016; 17: 1229

[15] Martinez-Alonso A, Nicolas-Espinosa J, Carvajal M. et al. The differential expressions of aquaporins underline the diverse strategies of cucumber and tomato against salinity and zinc stress. Physiol Plant. 2024; 176: e14222

[16] Jia Z, Yu W, Guo X. et al. CPK12 decodes effector-triggered calcium signaling and phosphorylates PIP2;1 to facilitate apoplastic ROS transport into the cytoplasm in Arabidopsis. Mol Plant. 2025; 18: 1724-41

[17] Quiroga G, Erice G, Aroca R. et al. Elucidating the possible involvement of maize aquaporins and arbuscular mycorrhizal symbiosis in the plant ammonium and urea transport under drought stress conditions. Plants (Basel). 2020; 9: 148

[18] Ludevid D, Höfte H, Himelblau E. et al. The expression pattern of the tonoplast intrinsic protein γ-TIP in Arabidopsis thaliana is correlated with cell enlargement 1. Plant Physiol. 1992; 100: 1633-9

[19] Peret B, Li G, Zhao J. et al. Auxin regulates aquaporin function to facilitate lateral root emergence. Nat Cell Biol. 2012; 14: 991-8

[20] Phillips AL, Huttly AK . Cloning of two gibberellin-regulated cDNAs from Arabidopsis thaliana by subtractive hybridization: expression of the tonoplast water channel, γ-TIP, is increased by GA3. Plant Mol Biol. 1994; 24: 603-15

[21] Mandlik R, Sharma S, Rout P. et al. Genome-wide identification and characterisation of aquaporins in Rosa chinensis. J Hortic Sci Biotechnol. 2024; 99: 311-25

[22] Ha STT, Nguyen TK, Lim JH . Effects of air-exposure time on water relations, longevity, and aquaporin-related gene expression of cut roses. Hortic Environ Biote. 2021; 62: 63-75

[23] Rasouli O, Ahmadi N, Behmanesh M. et al. Effects of BA and TDZ on postharvest quality and expression of laccase and aquaporin genes in cut rose ’Sparkle’. S Afr J Bot. 2015; 99: 75-9

[24] Zhang S, Feng M, Chen W. et al. In rose, transcription factor PTM balances growth and drought survival via PIP2;1 aquaporin. Nat Plants. 2019; 5: 290-9

[25] Ma N, Xue J, Li Y. et al. Rh-PIP2;1, a rose aquaporin gene, is involved in ethylene-regulated petal expansion. Plant Physiol. 2008; 148: 894-907

[26] Chen W, Yin X, Wang L. et al. Involvement of rose aquaporin RhPIP1;1 in ethylene-regulated petal expansion through interaction with RhPIP2;1. Plant Mol Biol. 2013; 83: 219-33

[27] Li R, Yao J, Ming Y. et al. Integrated proteomic analysis reveals interactions between phosphorylation and ubiquitination in rose response to Botrytis infection. Hortic Res. 2024; 11: uhad238

[28] Chalvin C, Drevensek S, Dron M. et al. Genetic control of glandular trichome development. Trends Plant Sci. 2020; 25: 477-87

[29] Wu M, Bian XX, Hu SR. et al. A gradient of the HD-zip regulator woolly regulates multicellular trichome morphogenesis in tomato. Plant Cell. 2024; 36: 2375-92

[30] Zhou NN, Simonneau F, Thouroude T. et al. Morphological studies of rose prickles provide new insights. Hortic Res. 2021; 8: 221

[31] Zhou NN, Tang KX, Jeauffre J. et al. Genetic determinism of prickles in rose. Theor Appl Genet. 2020; 133: 3017-35

[32] Wang J, Chu Y, Yuan X. et al. A CAPRICE gene of Rosa rugosa (RrCPC) suppresses the trichome formation of Arabidopsis. Ind Crop Prod. 2023; 194: 116340

[33] Satterlee JW, Alonso D, Gramazio P. et al. Convergent evolution of plant prickles by repeated gene co-option over deep time. Science. 2024; 385: eado1663

[34] Zhang Y, Zhao M, Zhu W. et al. Nonglandular prickle formation is associated with development and secondary metabolism-related genes in Rosa multiflora. Physiol Plant. 2021; 173: 1147-62

[35] Zhong M-C, Jiang XD, Yang GQ. et al. Rose without prickle: genomic insights linked to moisture adaptation. Natl Sci Rev. 2021; 8: nwab092

[36] Mutte SK, Kato H, Rothfels C. et al. Origin and evolution of the nuclear auxin response system. eLife. 2018; 7: e33399

[37] van Dop M, Fiedler M, Mutte S. et al. DIX domain polymerization drives assembly of plant cell polarity complexes. Cell. 2020; 180: 427-439.e12

[38] Li Q, Tong T, Jiang W. et al. Highly conserved evolution of aquaporin PIPs and TIPs confers their crucial contribution to flowering process in plants. Front Plant Sci. 2022; 12: 761713

[39] Zardoya R, Ding X, Kitagawa Y. et al. Origin of plant glycerol transporters by horizontal gene transfer and functional recruitment. Proc Natl Acad Sci. 2002; 99: 14893-6

[40] Zhang L, Morales-Briones DF, Li Y. et al. Phylogenomics insights into gene evolution, rapid species diversification, and morphological innovation of the apple tribe (Maleae, Rosaceae). New Phytol. 2023; 240: 2102-20

[41] Zhang Y, Qian K, Yu Q. et al. Subgenomic divergence and functional innovation following whole-genome duplication in Maleae species of Rosaceae. Plant J. 2025; 123: e70499

[42] Zhang Z, Yang T, Liu Y. et al. Haplotype-resolved genome assembly and resequencing provide insights into the origin and breeding of modern rose. Nat Plants. 2024; 10: 1659-71

[43] Tian J, Pei H, Zhang S. et al. TRV-GFP: a modified tobacco rattle virus vector for efficient and visualizable analysis of gene function. J Exp Bot. 2014; 65: 311-22

[44] Figueiredo YG, Gasparini K, Bulut M. et al. The genetic basis of prickle loss in the Solanaceae. Trends Plant Sci. 2025; 30: 119-21

[45] Li X, Li F, Wang G. et al. LcMYB306 regulates litchi fruit water loss and browning by inhibiting the expression of LcPIP2;4. Hortic Res. 2025; 13: uhaf322

[46] Wan Q, Li Y, Cheng J. et al. Two aquaporins, PIP1;1 and PIP2;1, mediate the uptake of neonicotinoid pesticides in plants. Plant Commun. 2024; 5: 100830

[47] Mukherjee S, Roy S, Corpas FJ . Aquaporins: a vital nexus in H₂O₂-gasotransmitter signaling . Trends Plant Sci. 2024; 29: 681-93

[48] Zhu H, Bao Y, Peng H. et al. Phosphorylation of PIP2;7 by CPK28 or Phytophthora kinase effectors dampens pattern-triggered immunity in Arabidopsis. Plant Commun. 2025; 6: 101135

[49] Jing W, Li Y, Zhang S. et al. Aquaporin, beyond a transporter. Hortic Plant J. 2023; 9: 29-34

[50] Bezerra-Neto JP, de Araújo FC, Ferreira-Neto JRC. et al. Plant aquaporins: diversity, evolution and biotechnological applications. Curr Protein Pept Sci. 2019; 20: 368-95

[51] Fawcett JA, Maere S, Van de Peer Y . Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. Proc Natl Acad Sci USA. 2009; 106: 5737-42

[52] Finn RN, Cerdà J . Evolution and functional diversity of aquaporins. Biol Bull. 2015; 229: 6-23

[53] Soto G, Alleva K, Amodeo G. et al. New insight into the evolution of aquaporins from flowering plants and vertebrates: orthologous identification and functional transfer is possible. Gene. 2012; 503: 165-76

[54] Hussain A, Tanveer R, Mustafa G. et al. Comparative phylogenetic analysis of aquaporins provides insight into the gene family expansion and evolution in plants and their role in drought tolerant and susceptible chickpea cultivars. Genomics. 2020; 112: 263-75

[55] Wang Y, Zhao Z, Liu F. et al. Versatile roles of aquaporins in plant growth and development. Int J Mol Sci. 2020; 21: 9485

[56] Login FH, Nejsum LN . Aquaporin water channels: roles beyond renal water handling. Nat Rev Nephrol. 2023; 19: 604-18

[57] Ishibashi K, Sasaki S . Aquaporin water channels in mammals. Clin Exper Neph. 1997; 1: 247-53

[58] Gala HP, Lanctot A, Jean-Baptiste K. et al. A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana. Plant Cell. 2021; 33: 2197-220

[59] Zhang TQ, Xu ZG, Shang GD. et al. A single-cell RNA sequencing profiles the developmental landscape of Arabidopsis root. Mol Plant. 2019; 12: 648-60

[60] Ding L, Milhiet T, Couvreur V. et al. Modification of the expression of the aquaporin ZmPIP2;5 affects water relations and plant growth. Plant Physiol. 2020; 182: 2154-65

[61] Ding L, Milhiet T, Parent B. et al. The plasma membrane aquaporin ZmPIP2;5 enhances the sensitivity of stomatal closure to water deficit. Plant Cell Environ. 2022; 45: 1146-56

[62] Parent B, Hachez C, Redondo E. et al. Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: a trans-scale approach. Plant Physiol. 2009; 149: 2000-12

[63] Huang X, Yi P, Liu Y. et al. RrTTG1 promotes fruit prickle development through an MBW complex in Rosa roxburghii. Front Plant Sci. 2022; 13: 939270

[64] Zhu W, Zhang Z, Wu J. et al. Transcriptome analysis reveals insights into regulatory networks of prickle formation in Rosa multiflora and the role of RmNAC43 in lignin biosynthesis during prickle hardening. Int J Biol Macromol. 2025; 319: 145515

[65] Tang N, Cao Z, Wu P. et al. Comparative transcriptome analysis reveals hormone, transcriptional and epigenetic regulation involved in prickle formation in Zanthoxylum armatum. Gene. 2023; 871: 147434

[66] Tang N, Wu P, Cao Z. et al. A NAC transcription factor ZaNAC93 confers floral initiation, fruit development, and prickle formation in Zanthoxylum armatum. Plant Physiol Biochem. 2023; 201: 107813

[67] Sánchez-Romera B, Ruiz-Lozano JM, Li G. et al. Enhancement of root hydraulic conductivity by methyl jasmonate and the role of calcium and abscisic acid in this process. Plant Cell Environ. 2014; 37: 995-1008

[68] Calvo-Polanco M, Armada E, Zamarreño AM. et al. Local root ABA/cytokinin status and aquaporins regulate poplar responses to mild drought stress independently of the ectomycorrhizal fungus Laccaria bicolor. J Exp Bot. 2019; 70: 6437-46

[69] Feng S, Liu Z, Chen H. et al. PHGD: an integrative and user-friendly database for plant hormone-related genes. iMeta. 2024; 3: e164

[70] Wu T, Liu Z, Yu T. et al. Flowering genes identification, network analysis, and database construction for 837 plants. Hortic Res. 2024; 11: uhae013

[71] Khan MR, Hu JY, Riss S. et al. MPF2-like-a MADS-box genes control the inflated calyx syndrome in Withania (Solanaceae): roles of Darwinian selection. Mol Biol Evol. 2009; 26: 2463-73

[72] Briscoe A, Bybee SM, Bernard GD. et al. Positive selection of a duplicated UV-sensitive visual pigment coincides with wing pigment evolution in Heliconius butterflies. Proc Natl Acad Sci USA. 2010; 107: 3628-33

[73] Yu D, Dong X, Zou K. et al. A hidden mutation in the seventh WD40-repeat of COP1 determines the early flowering trait in a set of Arabidopsis myc mutants. Plant Cell. 2023; 35: 345-50

[74] Bao Y, Liu G, Shi X. et al. Primary and repetitive secondary somatic embryogenesis in Rosa hybrida ‘Samantha’. Plant Cell Tissue Organ Cult. 2012; 109: 411-8

[75] Buchfink B, Reuter K, Drost H-G . Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods. 2021; 18: 366-8

[76] Lu S, Wang J, Chitsaz F. et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020; 48: D265-8

[77] Katoh K, Kuma K, Toh H. et al. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005; 33: 511-8

[78] Nguyen LT, Schmidt HA, von Haeseler A. et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32: 268-74

[79] Letunic I, Bork P . Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021; 49: W293-6

[80] Jung S, Lee T, Cheng CH. et al. 15 years of GDR: new data and functionality in the genome database for Rosaceae. Nucleic Acids Res. 2019; 47: D1137-45

[81] Liu Z, Zhang C, He J. et al. plantGIR: a genomic database of plants. Hortic Res. 2024; 11: uhae342

[82] Chen C, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13: 1194-202

[83] Artimo P, Jonnalagedda M, Arnold K. et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012; 40: W597-603

[84] Blom N, Gammeltoft S, Brunak S . Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol. 1999; 294: 1351-62

[85] Kumar S, Stecher G, Tamura K . MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33: 1870-4

[86] Hu BH, Jin J, Guo AY. et al. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2014; 31: 1296-7

[87] Tang H, Krishnakumar V, Zeng X. et al. JCVI: a versatile toolkit for comparative genomics analysis. iMeta. 2024; 3: e211

[88] Yang Z . PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24: 1586-91

[89] Che DD, Zhang XY, Zhang JZ. et al. A review of the quantitative trait loci in Rosa. Acta Horticulturae Sinica. 2016; 43: 1765-75

[90] Guo M, Bi G, Wang H. et al. Genomes of autotetraploid wild and cultivated Ziziphus mauritiana reveal polyploid evolution and crop domestication. Plant Physiol. 2024; 196: 2701-20

[91] Kiełbasa SM, Wan R, Sato K. et al. Adaptive seeds tame genomic sequence comparison. Genome Res. 2011; 21: 487-93

[92] Lescot M, Déhais P, Thijs G. et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; 30: 325-7

[93] Database Resources of the National Genomics Data Center. China National Center for bioinformation in 2024. Nucleic Acids Res. 2024; 52: D18-d32

[94] Liu Y, Schiff M, Marathe R. et al. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 2002; 30: 415-29

[95] Schmittgen TD, Livak KJ . Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3: 1101-8

[96] Dong X, Zhang LP, Tang YH. et al. Arabidopsis AGAMOUS-LIKE16 and SUPPRESSOR OF CONSTANS1 regulate the genome-wide expression and flowering time. Plant Physiol. 2023; 192: 154-69