Nodulin 26-like Intrinsic Proteins Research Articles

Aquaporins: Multifunctional Players in Plant Growth, Development and Stress Responses

Aquaporins are small membrane proteins belonging to the group of Major Intrinsic proteins (MIPs) that facilitate the transport of water and small molecules across biological membranes. They were initially identified as water channels and later it has become clear that they also play important roles in several other physiological processes in plants. Plants have different types of aquaporins in terms of their location, gating properties, and solute specificity and can mainly divide into four major subfamilies. They are tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), plasma membrane intrinsic proteins (PIPs), and the small basic intrinsic proteins (SIPs). All these aquaporins play a variety of physiological activities at all stages of plant growth and development. Also, they are believed to play a significant part in the plant’s defense mechanisms against biotic and abiotic stresses. This review comprehensively addresses the roles of diverse aquaporin homologs in growth and development and their response to different environmental factors.

Boron homeostasis affects Longan yield: a study of NIP and BOR boron transporter of two cultivars

BackgroundEssential micronutrient Boron (B) plays crucial roles in plant survival and reproduction but becomes toxic in higher quantities. Although plant cells have different B transport systems, B homeostasis is mainly maintained by two transporter protein families: B exporters (BOR) and nodulin-26-like intrinsic proteins (NIP). Their diversity and differential expression are responsible for varied B tolerance among plant varieties and species. Longan is a highly admired subtropical fruit with a rising market in China and beyond. In the present study, we cultured Shixia (SX) and Yiduo (YD), two differently characterized Longan cultivars, with foliar B spray. We analyzed their leaf physiology, fruit setting, B content, and boron transporter gene expression of various tissue samples. We also traced some of these genes' subcellular localization and overexpression effects.ResultsYD and SX foliage share similar microstructures, except the mesophyll cell wall thickness is double in YD. The B spray differently influenced their cellular constituents and growth regulators. Gene expression analysis showed reduced BOR genes expression and NIP genes differential spatiotemporal expression. Using green fluorescent protein, two high-expressing NIPs, NIP1 and NIP19, were found to translocate in the transformed tobacco leaves' cell membrane. NIPs transformation of SX pollen was confirmed using magnetic beads and quantified using a fluorescence microscope and polymerase chain reaction. An increased seed-setting rate was observed when YD was pollinated using these pollens. Between the DlNIP1 and DlNIP19 transformed SX pollen, the former germinated better with increasing B concentrations and, compared to naturally pollinated plants, had a better seed-setting rate in YD♀ × SX♂.ConclusionSX and YD Longan have different cell wall structures and react differently to foliar B spray, indicating distinct B tolerance and management. Two B transporter NIP genes were traced to localize in the plasma membrane. However, under high B concentrations, their differential expression resulted in differences in Jasmonic acid content, leading to differences in germination rate. Pollination of YD using these NIPs transformed SX pollen also showed NIP1 overexpression might overcome the unilateral cross incompatibility between YD♀ × SX♂ and can be used to increase Longan production.

Mechanistic Basis of Silicon Mediated Cold Stress Tolerance in Alfalfa (Medicago sativa L.)

AbstractCold stress (CS) impact on crops is one of the critical constraints for sustainable and smart agricultural production. CS adversely affects plants leading to growth retardation, necrosis, chlorosis, and significant yield loss. The objective of this study was to explore the mechanistic basis of silicon (Si) in enhancing CS tolerance in alfalfa plants. The fluorescence staining indicated that Si-reduced the intensity of CS-induced superoxide radical (O2•–) and hydrogen peroxide (H2O2) generation in plants that improved plant photosynthesis, cellular integrity, and alfalfa biomass production under CS. The exogenous supplementation of Si significantly restored the endogenous Si status accompanied by the upregulation of NIP (nodulin 26-like intrinsic protein) genes NIP2, NIP5;1, and NIP6;1 in alfalfa. The elemental concentration analysis revealed that exogenous silicon (E-Si) triggers the increase of calcium (Ca), magnesium (Mg), and sulfur (S) in plants subjected to Si-supplementation compared to the plants cultivated without Si under CS. The application of Si significantly increased the activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Furthermore, Si significantly enhanced the expression of CS-responsive candidate genes including ICE1, CBF1/DREB1C, CBF2/DREB1B, CBF3/DREB1A, COR15A, COR47, and KIN1 in alfalfa. These findings together provide mechanistic insights into Si-involving CS tolerance in alfalfa. This eco-friendly SC management strategy using Si treatment can be useful to plant breeders and farmers for developing CS-resilient smart alfalfa production through breeding program.

Barley Nodulin 26-like intrinsic protein permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification

Aquaporins can facilitate the passive movement of water, small polar molecules and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2,993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.

Identification of VrNIP2-1 aquaporin with novel selective filter regulating the transport of beneficial as well as hazardous metalloids in mungbean (Vigna radiata L.)

Nodulin 26-like intrinsic protein (NIP) subfamily of aquaporins (AQPs) in plants, is known to be involved in the uptake of metalloids including boron, germanium (Ge), arsenic (As), and silicon (Si). In the present study, a thorough evaluation of 55 AQPs found in the mungbean genome, including phylogenetic distribution, sequence homology, expression profiling, and structural characterization, contributed to the identification of VrNIP2-1 as a metalloid transporter. The pore-morphology of VrNIP2-1 was studied using molecular dynamics simulation. Interestingly, VrNIP2-1 was found to harbor an aromatic/arginine (ar/R) selectivity filter formed with ASGR amino acids instead of GSGR systematically reported in metalloid transporters (NIP2s) in higher plants. Evaluation of diverse cultivars showed a high level of Si accumulation in leaves indicating functional Si transport in mungbean. In addition, heterologous expression of VrNIP2-1 in yeast revealed As(III) and GeO2 transport activity. Similarly, VrNIP2-1 expression in Xenopus oocytes confirmed its Si transport ability. The metalloid transport activity with unique structural features will be helpful to better understand the solute specificity of NIP2s in mungbean and related pulses. The information provided here will also serve as a basis to improve Si uptake while restricting hazardous metalloids like As in plants.

Genome-Wide Analysis of Aquaporins in Japanese Morning Glory (Ipomoea nil)

The aquaporin (AQP) family, also called water channels or major intrinsic proteins, facilitate water transport. AQPs also transport low-molecular-weight solutes, including boric acid, glycerol, urea, and ammonia. Since plants are sessile, water homeostasis is crucial. Therefore, plants have developed diverse AQP variants at higher expression levels than animals. For example, 35 and 33 AQPs have been identified in Arabidopsis and rice, respectively. In the present study, we identified AQPs in morning glory (Ipomoea nil), which has been widely used as a model plant in research on flowering and floral morphology. The importance of AQPs in the opening of morning glory flowers has been reported. In the morning glory genome, 44 AQPs were identified, and their characteristics were analyzed. A phylogenetic analysis revealed five AQP subfamilies in morning glory: plasma membrane-intrinsic proteins (PIPs), tonoplast-intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs), and X-intrinsic proteins (XIPs). Further, transport substrates of morning glory AQPs were estimated based on their homology to the known AQPs in other plant species and their corresponding amino acid motifs that possess permeability pores. It was expected that PIPs are likely to transport water, carbon dioxide, and hydrogen peroxide; TIPs are likely transport water, hydrogen peroxide, ammonia, urea, and boric acid; NIPs are likely transport water, boric acid, ammonia, glycerol, and formamide; and XIPs are likely to transport water, hydrogen peroxide, and glycerol. Overall, these results suggest that AQPs are involved in water and nutrient transport in Japanese morning glory. An in silico gene expression analysis suggested the importance of AQPs in flower opening, water or nutrient uptakes from the soil to roots, and photosynthesis in morning glory. Our findings provide fundamental information that enables further study into the importance of AQPs in morning glory, including their roles in flower opening and other physiological events.

Boric acid transport activity of marine teleost aquaporins expressed in Xenopus oocytes.

Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin-like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.

Research advances on the structure, function and regulation of NIP aquaporins in plants

<p indent="0mm">Aquaporin protein (AQP) family, as a “carrier” of water, micronutrients, and solute macromolecules in plants, plays an important role in material transport. Unlike other members of the AQP family, nodulin 26-like intrinsic proteins (NIPs) mainly penetrate small molecules, such as water, glycerin, silicic acid, boric acid, and urea. In addition, it is involved in plant cell osmoregulation, seed germination, lateral root production, leaf and flower growth and development, and response to biotic and abiotic (drought, chilling, salinity, metalloid toxicity, hypoxia, etc.) stresses. So far, structures of the NIP family have been partially reported. The NIPs can be divided into three classes based on the pore size of the ar/R selective filter, which determines the absorption and transportation of different substrates. Moreover, the NIPs contain two NPA motifs, which are located in loop B and loop E and have a typical ring-like hourglass structure. Therefore, different structures of the NIP subfamily play different roles in plant membrane solute transport and water balance. The <italic>NIP</italic> gene family has been identified in <italic>Arabidopsis thaliana</italic>,<italic> Oryza sativa</italic>, and other plants, whose function needs to be achieved through a variety of signaling pathways and the synergistic effect of physiological processes. In recent years, more and more reports have shown how the <italic>NIP</italic> gene participates in regulating gene expression levels under various stress conditions and interacts with a variety of stress proteins to jointly regulate the water and osmotic balance of plants and improve plant resistance and adaptability. The co-expression of <italic>NIPs</italic> with <italic>AtBOR1</italic>, <italic>AtNodGS</italic>, and <italic>AtACR3</italic> genes can enhance the transport of solute and aquaporin. Besides, the interaction between NIP and 14-3-3f proteins can stabilize cell structure and substance synthesis in vivo, thus enhancing plant cold resistance. Interestingly, <italic>NIP</italic> is regulated by WRKY transcription factors and plays an active role in the regulation and distribution of metalloid elements in plants. In addition, the <italic>NIP </italic>genes are involved in the response of various hormone signaling pathways in plants, and their gating mechanisms and osmotic stress responses are regulated by the C- and N-terminal phosphorylation sites of NIP proteins, but the specific response mechanisms remain to be further explored. <italic>NIPs</italic> are a large multi-gene family with functional redundancy among members, and the similarities and differences in functions and mechanisms of different subfamilies of <italic>NIP</italic> genes need to be investigated. One of our main subsequent research directions is to explore the stress resistance network of the <italic>NIP</italic> genes, especially their response mechanisms to biotic stress. What’s more, NIPs can interact with PIP proteins belonging to the AQP family and affect the diffusion of water molecules by changing their positions. Therefore, whether NIPs interact with other members of the AQP family, such as SIPs and TIPs, deserves to be validated further. In summary, the origin and evolution, structure, classification, biological function, and regulation mechanism of the NIP family in plants are reviewed, which provides new insights and references for further research.

Genome-wide identification and biological relevance of broccoli aquaporins.

Broccoli (Brassica oleracea var. italica) is an important crop worldwide, and its regular consumption is associated with health benefits due to the presence of various bioactive compounds. An optimal water balance and homeostasis are needed for plant growth; in this sense, aquaporins play a crucial role. As a result of a genome-wide search, a total of 65 aquaporin genes were identified in broccoli. The aquaporins were classified according to their phylogenetic relationships with other Brassicas species and Arabidopsis thaliana, and evolutionary events of gene duplication were also assessed, highlighting the tendency of NIPs (Nodulin-26-like Intrinsic Proteins) to duplicate. Also, the chromosomal localization, gene duplication, the study of the conserved motifs, and the tertiary structure were determined in broccoli. Functional predictive analyses were also carried out, which, together with the expression analyses in different broccoli plant tissues, allowed the prediction of the biological role of each aquaporin isoform. BoiPIP1-2a and BoiPIP1-2b showed higher expression in all the plant tissues when compared with other aquaporins. BoiTIP1-2b also showed high expression levels and was associated with nitrogen compounds transport such as urea. However, NIPs, through their differential expression and the tandem duplications of the isoforms, were revealed as the putative main actors in the response of broccoli plants to abiotic stress responses. The results of this work pointed to the physiological significance of each aquaporin isoform of broccoli, opening a new field of knowledge and constituting the first step of further in vivo analyses.

The plasma membrane-localized OsNIP1;2 mediates internal aluminum detoxification in rice.

Aluminum (Al) toxicity significantly restricts crop production on acidic soils. Although rice is highly resistant to Al stress, the underlying resistant mechanisms are not fully understood. Here, we characterized the function of OsNIP1;2, a plasma membrane-localized nodulin 26-like intrinsic protein (NIP) in rice. Aluminum stress specifically and quickly induced OsNIP1;2 expression in the root. Functional mutations of OsNIP1;2 in two independent rice lines led to significantly enhanced sensitivity to Al but not other metals. Moreover, the Osnip1;2 mutants had considerably more Al accumulated in the root cell wall but less in the cytosol than the wild-type rice. In addition, compared with the wild-type rice plants, the Osnip1;2 mutants contained more Al in the root but less in the shoot. When expressed in yeast, OsNIP1;2 led to enhanced Al accumulation in the cells and enhanced sensitivity to Al stress, suggesting that OsNIP1;2 facilitated Al uptake in yeast. These results suggest that OsNIP1;2 confers internal Al detoxification via taking out the root cell wall’s Al, sequestering it to the root cell’s vacuole, and re-distributing it to the above-ground tissues.

Involvement of NGATHA-Like 1 Transcription Factor in Boron Transport under Low and High Boron Conditions.

NGATHA-Like 1 (NGAL1) transcription factor has been identified as a gene regulated through AUG-stop-mediated boron (B)-dependent translation stall; however, its function in B response remains unknown. Here, we show that NGAL1 plays an important role in the maintenance of B transport under both low- and high-B conditions in Arabidopsis thaliana. NGAL1 mRNA is accumulated predominantly in shoots in response to B stress. Independent ngal1 mutants carrying transferred DNA (T-DNA) and Ds-transposon insertions exhibit reduced B concentrations in aerial tissues and produce shortened and reduced number of siliques when B supply is limited. Furthermore, the expression of B transporter genes including nodulin 26-like intrinsic protein 6; 1 (NIP6;1), NIP5;1, NIP7;1 and borate exporter 1 (BOR1) is significantly decreased in ngal1 mutants under low-B condition, suggesting that NGAL1 is required for the transcript accumulation of B transporter genes to facilitate B transport and distribution under B limitation. Under high-B condition, ngal1 mutants exhibit reduced growth and increased B concentration in their shoots. The accumulation of BOR4 mRNA, a B transporter required for B efflux to soil, is significantly reduced in roots of ngal1 plants under high-B condition, suggesting that NGAL1 is involved in the upregulation of BOR4 in response to excess B. Together, our results indicate that NGAL1 is involved in the transcriptional regulation of B transporter genes to facilitate B transport and distribution under both low- and high-B conditions.

The structure, function and expression analysis of the nodulin 26-like intrinsic protein subfamily of plant aquaporins in tomato

The nodulin 26-like intrinsic protein (NIP) family belonging to a group of aquaporin proteins is unique to plants. NIPs have a wide of transport activities and are involved in developmental processes and stress tolerance. The well reported Lsi1 and Lsi6 belonging to NIP III were characterized as Si transporters. However, except Lsi1 and Lsi6, most NIPs remain unknown. Here, we identified 43 putative aquaporins in tomato. We found there are 12 NIPs, including 8 NIP I proteins, 3 NIP II proteins, and 1 NIP III protein among the 43 aquaporins. Also, there are two Si efflux transporters SlLsi2-1 and SlLsi2-2 identified by using Lsi2 proteins from other species. By analysing the phylogenetic relationships, conserved residues and expression patterns, we propose that three NIP I members (SlNIP-2, SlNIP-3 and SlNIP-11) may transport water, ammonia, urea, and boric acid, and contribute to pollen development. Three NIP II proteins (SlNIP-7, SlNIP-9 and SlNIP-12) may be boric acid facilitators, and affect plant growth and anther development. Overall, the study provides valuable candidates of Si transporters and other NIP proteins to further explore their roles in uptake and transport for silicon, boron, and other substrates in tomato.

Pearl Millet Aquaporin Gene PgPIP2;6 Improves Abiotic Stress Tolerance in Transgenic Tobacco.

Pearl millet [Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T2 generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops.

Two Aquaporin Genes, GhPIP2;7 and GhTIP2;1, Positively Regulate the Tolerance of Upland Cotton to Salt and Osmotic Stresses.

Aquaporins (AQPs) facilitate the transport of water and small molecules across intrinsic membranes and play a critical role in abiotic stresses. In this study, 111, 54, and 56 candidate AQP genes were identified in Gossypium hirsutum (AD1), Gossypium arboreum (A2), and Gossypium raimondii (D5), respectively, and were further classified into five subfamilies, namely, plasma intrinsic protein (PIP), tonoplast intrinsic protein (TIP), nodulin 26-like intrinsic protein (NIP), small basic intrinsic protein (SIP), and uncategorized X intrinsic protein (XIP). Transcriptome analysis and quantitative real-time PCR (qRT-PCR) revealed some high-expression GhPIPs and GhTIPs (PIP and TIP genes in G. hirsutum, respectively) in drought and salt stresses. GhPIP2;7-silenced plants decreased in the chlorophyll content, superoxide dismutase (SOD) activity, and peroxidase (POD) activity comparing the mock control (empty-vector) under 400 mM NaCl treatment, which indicated a positive regulatory role of GhPIP2;7 in salt tolerance of cotton. The GhTIP2;1-silenced cotton plants were more sensitive to osmotic stress. GhTIP2;1-overexpressed plants exhibited less accumulation of H2O2 and malondialdehyde but higher proline content under osmotic stress. In summary, our study elucidates the positive regulatory roles of two GhAQPs (GhPIP2;7 and GhTIP2;1) in salt and osmotic stress responses, respectively, and provides a new gene resource for future research.

Nodulin 26-like intrinsic protein CsNIP2;2 is a silicon influx transporter in Cucumis sativus L.

Nodulin 26-like intrinsic proteins (NIPs) are a family of channel-forming transmembrane proteins that function in the transport of water and other small molecules. Some NIPs can mediate silicon transport across plasma membranes and lead to silicon accumulation in plants, which is beneficial for the growth and development of plants. Cucumber is one of the most widely consumed vegetables; however, the functions of NIPs in this crop are still largely unknown. Here, we report the functional characteristics of CsNIP2;2. It was found that CsNIP2;2 is a tandem repeat of CsNIP2;1, which had been demonstrated to be a silicon influx transporter gene. CsNIP2;2 has a selectivity filter composed of cysteine, serine, glycine and arginine (CSGR), which is different from all previously characterized silicon influx transporters in higher plants at the second helix position. Xenopus laevis oocytes injected with CsNIP2;2 cRNA demonstrated a higher uptake of silicon than the control, and the uptake remained unchanged under low temperature. CsNIP2;2 was found to be expressed in the root, stem, lamina and petiole, and exogenous silicon treatment decreased its expression in the stem but not in other tissues. Transient expression of CsNIP2;2-eGFP fusion sequence in onion epidermal cells showed that CsNIP2;2 was localized to the cell nucleus, plasma membrane and an unknown structure inside the cell. The results suggest that CsNIP2;2 is a silicon influx transporter in cucumber, and its subcellular localization and the selectivity filter are different from those of the previously characterized silicon influx transporters in other plants. These findings may be helpful for understanding the functions of NIPs in cucumber plants.

The roles of selectivity filters in determining aluminum transport by AtNIP1;2

ABSTRACT Aquaporins (AQPs) are channel proteins involved in transporting a variety of substrates. It has been proposed that the constriction regions in the central pores of the AQP channels play a crucial role in determining transport substrates and activities of AQPs. Our previous results suggest that AtNIP1;2, a member of the AQP superfamily in Arabidopsis, facilitates aluminum transport across the plasma membrane. However, the functions of the constriction regions in AtNIP1;2-mediated transport activities are unclear. This study reports that residue substitutions of the constriction regions affect AtNIP1;2-mediated aluminum uptake, demonstrating the critical roles of the constriction regions for transport activities. Furthermore, a constriction region that partially or wholly mimics AtNIP5;1, a demonstrated boric-acid transporter, could not render the boric-acid transport activity to AtNIP1;2. Therefore, besides the constriction regions, other structural features are also involved in determining the nature of AtNIP1;2’s transport activities. Abbreviations: AIAR: alanine-isoleucine-alanine-arginine; AIGR: alanine-isoleucine-glycine- arginine; AQP: aquaporin; Al-Mal: aluminum-malate; ar/R: aromatic/arginine; AVAR: alanine-valine-alanine-arginine; CK: control; H: helical domain; ICP-MS: inductively coupled plasma mass spectrometry; LA - LE: inter-helical loops A to E; NIP: nodulin 26-like intrinsic protein; NPA: asparagine-proline-alanine; NPG: asparagine-proline- glycine; NPS: asparagine-proline-Serine; NPV: asparagine-proline-valine; ORF: open reading frame; PIP: plasma membrane intrinsic proteins; SIP: small basic intrinsic proteins; TM: transmembrane helices; WIAR: tryptophan-isoleucine-alanine-arginine; WVAR: tryptophan-valine-alanine-arginine; WVGR: tryptophan-valine-glycine- arginine.

Structural basis for high selectivity of a rice silicon channel Lsi1

Silicon (Si), the most abundant mineral element in the earth’s crust, is taken up by plant roots in the form of silicic acid through Low silicon rice 1 (Lsi1). Lsi1 belongs to the Nodulin 26-like intrinsic protein subfamily in aquaporin and shows high selectivity for silicic acid. To uncover the structural basis for this high selectivity, here we show the crystal structure of the rice Lsi1 at a resolution of 1.8 Å. The structure reveals transmembrane helical orientations different from other aquaporins, characterized by a unique, widely opened, and hydrophilic selectivity filter (SF) composed of five residues. Our structural, functional, and theoretical investigations provide a solid structural basis for the Si uptake mechanism in plants, which will contribute to secure and sustainable rice production by manipulating Lsi1 selectivity for different metalloids.

Transcriptome analysis reveals the molecular mechanism of boron deficiency tolerance in leaves of boron-efficient Beta vulgaris seedlings

Sugar beet (Beta vulgaris L.) has a high demand for B, and B deficiency inhibits normal growth and productivity. However, there is a lack of information on how B deficiency affects the growth of beet at the transcriptome level, and the factors that govern B utilisation efficiency. This study aimed to identify the genes differentially expressed under B deficiency and those that underlie the mechanisms of efficient B use in two sugar beet cultivars. Accordingly, B-efficient (H, KWS1197) and B-inefficient (L, KWS0143) sugar beet cultivars were used, and two levels of boron were employed in the hydroponic experiments: B0.1 (0.1 μM B, deficiency) and B50 (50 μM B, CK). The results showed that B deficiency inhibited leaf growth, significantly reduced B concentration and B transfer coefficient, and increased peroxidase (POD) activity and malondialdehyde and proline content. The transcriptome data showed that the B-efficient variety exhibited more differentially expressed genes than the B-inefficient variety. Metabolic pathways were the most critical pathways involved in the B deficiency response. The expression of POD, bHLH, WRKY transcription factors, and nodulin26-like intrinsic protein (NIP5;1) were upregulated in the KWS1197 variety. In conclusion, the KWS1197 variety had physiological advantages and a highly efficient B utilisation molecular mechanism, contributing to a high B deficiency tolerance. This study provides a theoretical basis for the adaptation mechanism to B deficiency in sugar beets.

Aquaporin family lactic acid channel NIP2;1 promotes plant survival under low oxygen stress in Arabidopsis.

Under anaerobic stress, Arabidopsis thaliana induces the expression of a collection of core hypoxia genes that encode proteins for an adaptive response. Among these genes is NIP2;1, which encodes a member of the "Nodulin 26-like Intrinsic Protein" (NIP) subgroup of the aquaporin superfamily of membrane channel proteins. NIP2;1 expression is limited to the "anoxia core" region of the root stele under normal growth conditions, but shows substantial induction (up to 1,000-fold by 2-4 h of hypoxia) by low oxygen stress, and accumulation in all root tissues. During hypoxia, NIP2;1-GFP accumulates predominantly on the plasma membrane by 2 h, is distributed between the plasma and internal membranes during sustained hypoxia, and remains elevated in root tissues through 4 h of reoxygenation recovery. In response to hypoxia challenge, T-DNA insertion mutant nip2;1 plants exhibit elevated lactic acid within root tissues, reduced efflux of lactic acid, and reduced acidification of the external medium compared to wild-type plants. Previous biochemical evidence demonstrates that NIP2;1 has lactic acid channel activity, and our work supports the hypothesis that NIP2;1 prevents lactic acid toxicity by facilitating release of cellular lactic acid from the cytosol to the apoplast, supporting eventual efflux to the rhizosphere. In evidence, nip2;1 plants demonstrate poorer survival during argon-induced hypoxia stress. Expressions of the ethanolic fermentation transcript Alcohol Dehydrogenase1 and the core hypoxia-induced transcript Alanine Aminotransferase1 are elevated in nip2;1, and expression of the Glycolate Oxidase3 transcript is reduced, suggesting NIP2;1 lactic acid efflux regulates other pyruvate and lactate metabolism pathways.

The NIP Genes in Sugar Beet: Underlying Roles in Silicon Uptake and Growth Improvement

Silicon (Si) can stimulate plant growth and stress resistance. This study aimed at elucidating the physiological responses and molecular characterization of different NIP (nodulin 26-like intrinsic protein) genes in sugar beet. The addition of exogenous Si increased plant biomass and Si concentration in the root and shoot, implying that increased Si uptake has beneficial effects on sugar beet. The quantitative gene expression analysis showed a significant upregulation of the BvNIP5–1 gene in roots, while the BvNIP1–1, BvNIP6–1, BvNIP7–1, and BvNIP-type were unchanged following Si supplementation. It suggests that BvNIP5–1 is one of the genes localized in the plasma membrane responsible for Si uptake in sugar beet. Besides, the BvNIP5–1 gene contained promoter, transcription start site, transcription binding site, and PolyA at 800 bp, 528 bp, 557 bp, and 1637 bp, respectively. The MEME results further revealed the association of three motifs related to major intrinsic protein (MIP). In the phylogenetic tree, the BvNIP5–1 showed a close association with CqNIP5–1, AnNIP2–1 and SoNIP5–1. Interactome analysis showed functional partnership of aquaporin SIP1–2 and SIP2–1 genes with NIP genes in sugar beet. In addition, BvNIP5–1 possessed close association with several functional partners (BOR1, BOR1, NodGS, SIP1A, ACR3) and co-expressed genes (i.e., AtNIP5–1, AtBOR1, AtNodGS, AtSIP1A, AtACR3) of Arabidopsis thaliana mainly responsible for boron, arsenite, and aquaporin transport activity. NIP promoters revealed cis-regulatory elements linked to auxin, salicylic acid, and methyl jasmonate-responsive factors. These findings may advance our understanding of Si transporters underlying growth and stress tolerance in sugar beet.