Arabidopsis NIP2;1, a Major Intrinsic Protein Transporter of Lactic Acid Induced by Anoxic Stress

Abstract

Nodulin 26 intrinsic proteins (NIPs) are plant-specific, highly conserved water and solute transport proteins with structural and functional homology to soybean nodulin 26. Arabidopsis thaliana contains nine NIP genes. In this study, it is shown that one of these, AtNIP2;1, is exquisitely sensitive to water logging and anoxia stress. Based on quantitative PCR and promoter::GUS experiments, AtNIP2;1 is expressed at a low basal level in the root tips and the vascular bundle of differentiated roots. Transcript levels are elevated acutely and rapidly upon water logging of root or leaf tissues, increasing 70-fold in roots within the 1st h of submersion. After this large initial increase, mRNA levels decline to steady state levels that remain over 10-fold higher by 6 h post-submersion. An even greater induction of AtNIP2;1 expression was observed upon anoxia challenge of Arabidopsis seedlings, with a 300-fold increase in AtNIP2;1 transcript observed by 2 h after the initiation of oxygen deprivation. Functional analysis of AtNIP2;1 expressed in Xenopus oocytes shows that the protein differs from soybean nodulin 26, showing minimal water and glycerol transport. Instead, AtNIP2;1 displays transport of lactic acid, with a preference for the protonated acidic form of this weak acid. Overall, the data suggest that AtNIP2;1 is an anaerobic-induced gene that encodes a lactic acid transporter and may play a role in adaptation to lactic fermentation under anaerobic stress.Major intrinsic proteins (MIPs) 2 are an ancient integral membrane channel protein family of water and uncharged solute transporters that have been found in nearly all living organisms. MIPs are especially prevalent and diverse in higher plants and have been classified into four monophyletic groups as follows: the plasma membrane intrinsic proteins (PIPs), the tonoplast intrinsic proteins, the nodulin 26-like intrinsic proteins (NIPs), and the small basic intrinsic proteins (1).NIPs are named for soybean nodulin 26 (Nod26) (2), which is the major protein component of the symbiosome membrane from nitrogen-fixing soybean root nodules (3, 4). Functional analyses indicate that Nod26 is an aquaglyceroporin with a low intrinsic water permeability and the ability to transport uncharged metabolites such as glycerol (4, 5) and has also been implicated in ammonia transport (6). It has become clear that NIPs represent a large, diverse family of aquaglyceroporins, with multiple members found in every sequenced higher plant genome (1,7,8). For example, among the 35 MIP genes in Arabidopsis thaliana (1), there are nine members of the NIP subfamily. Based on molecular modeling of the pore selectivity sequences, these nine NIPs are subdivided into the following two groups: NIP subgroup I proteins are encoded by NIP1;1, NIP1;2, NIP2;1, NIP3;1, NIP4;1, and NIP4;2, and NIP subgroup II proteins are encoded by NIP5;1, NIP6;1, and NIP7;1 (9).Analysis of NIP subgroup I proteins shows that they are more similar to soybean nodulin 26 in structure and function, with several showing aqu...

Genome-wide identification and characterization of aquaporin gene family in common bean (Phaseolus vulgaris L.)

“…This aquaporin sub-family has a wide range of substrate specificity such as water, glycerol, urea, boric acid, silicon, and lactic acid (Dean et al 1999;Wallace and Roberts 2005;Ma et al 2006;Takano et al 2006;Choi and Roberts 2007). This specificity is based mainly on sequence differences in the ar/R selectivity filter that are used also for dividing this sub-family in three distinct subgroups (Wallace and Roberts 2005).…”

Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress

“…Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 seeds were germinated and grown under a long-day cycle of 16 h of light (approximately 100 mmol m 22 s 21 )/8 h of dark at 22°C as described previously (Choi and Roberts, 2007). For hydroponic growth, 10-d-old seedlings were transferred onto a hydroponics growth system (Gibeaut et al, 1997) and were grown under long-day conditions.…”

“…GUS staining was performed on Arabidopsis seedlings as described by Choi and Roberts (2007). Epifluorescence images were captured with an Axiovert 200M microscope (Zeiss) equipped with filters for YFP fluorescence (Chroma; filter set 52017) and a digital camera (Hamamatsu Orca-ER) controlled by the Openlab software (Improvision).…”

“…Total RNA was isolated and Q-PCR analysis was performed as described previously (Choi and Roberts, 2007) with the gene-specific and internal control primers are described in Supplemental Table S3. The Arabidopsis UBQ10 gene was used as an internal reference for standardization, and quantitation of gene expression was calculated using the comparative threshold cycle method as described previously (Choi and Roberts, 2007).…”

“…The Arabidopsis UBQ10 gene was used as an internal reference for standardization, and quantitation of gene expression was calculated using the comparative threshold cycle method as described previously (Choi and Roberts, 2007). For end-point reverse transcription-PCR, 50 ng of 2-h hypoxic root cDNA from 12-d-old wild-type, CML38-1 complementation, and cml38 mutant seedlings was combined with 500 nM CML38 or ACTIN2 (internal reference) gene primer sets and 12.5 mL of the GoTaq Green Master Mix (Promega) in a final volume of 25 mL.…”