Rice aquaporin OsPIP2;2 is a water-transporting facilitator in relevance to drought-tolerant responses

Abstract

In rice (Oryza sativa), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H₂O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H₂O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIPs, four genes (OsPIP1;3, OsPIP2;1, OsPIP2;2, and OsPIP2;4) were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H₂O. In comparison, OsPIP2;2 was most supportive to H₂O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H₂O transport across the plasma membranes. OsPIP2;2-mediated H₂O transport was accompanied by drought-tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H₂O transport and drought-tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H₂O transport in relevance to drought tolerance in the plant.

Keywords: PIP2;2; aquaporin; drought tolerance; rice; water transport.

FIGURE 1

Chronological changes in rice response to physiological drought stress caused by PEG₆₀₀₀ applied in a range of concentration. Fifteen‐day‐old rice seedlings were incubated by root immersion in deionized water containing PEG₆₀₀₀ added at the indicated concentrations. Images were obtained by automatic photography in 10 h at 10‐min intervals and are partially shown here on the hour except one image in which seedlings incubated with the highest concentration of PEG₆₀₀₀ had displayed drought syndromes. Arrowheads point the concentrations that already induced drought syndromes. Each image represents 105 plants treated proportionally with each of the 7 PEG₆₀₀₀ doses and tested in 3 independent experiments

FIGURE 2

Expression levels of OsPIPs in rice plants temporally incubated in deionized water with and without supply with PEG₆₀₀₀. Rice seedlings already growing for 15 days in pot soil in a plant growth chamber were shifted into tubes containing deionized water. After 12‐h acclimation, these plants were transferred into new tubes containing deionized water only or an aqueous solution of PEG₆₀₀₀ at the indicated concentration. Insets show morphological changes of seedlings growing under the indicated conditions. Gene expression was analyzed by QRT‐PCR performed with total RNAs isolated from the aerial parts of the plants 3 h after incubation. The constitutively expressed OsEF1α gene was used as a reference to quantify relative expression level of an OsPIP. Data show are mean values ± standard deviation (SD) estimates. The number of experimental repeats (n) = 6 independent experiments each involving 15 plants tested in three biological repeats. Different letters on graphs indicate significant differences by analysis of variance (ANOVA) and Fisher's test between the pairs of data (P = .001–1.2 × 10⁻⁷)

FIGURE 3

De novo expression of OsPIP‐eGFPs in African clawed frog oocytes and the subsequent effects on osmotic water permeability (Pf) of the oocyte membranes. (a) Microscopic observations on oocytes 48 h after transformation with the recombinant vector containing OsPIP‐eGFP fusion genes or with the empty vector (control) that did contain any gene insert. Each image represents 60 oocytes transformed in 6 independent experiments. (b) Immunoblotting of oocyte proteins hybridized with the GFP antibody. (c) Pf measurements. Data shown are mean values ± SDs of results from 6 independent experiments each involving 10 oocytes (n = 60). Different letters on graphs indicate significant differences based on ANOVA and Duncan's multiple new multiple range test of the data obtained from the different oocytes (P =.056–1.5 × 10⁻¹⁶)

FIGURE 4

De novo expression of OsPIP‐His fusion proteins in African clawed frog oocytes and the subsequent effects on Pf of the oocyte membranes. (a) Immunoblotting of oocyte proteins hybridized with the His antibody. (b) Pf measurements. Data shown are mean values ± SDs of results from 6 independent experiments each involving 10 oocytes (n = 60). Different letters on graphs indicate significant differences based on ANOVA and Duncan's multiple new multiple range test of the data obtained from the different oocytes (P = .056–1.2 × 10⁻¹⁷)

FIGURE 5

De novo expression of OsPIP‐His fusion genes in yeast and the subsequent effects on Pf of the yeast membranes. (a) Relative levels of OsPIP expression in yeast cultures 24 h after transformation with the recombinant vector containing an insert of the OsPIP‐his fusion genes in comparison with the empty vector (control). (b) Quantification of OsPIP‐His fusion proteins based on immunoblotting of yeast proteins hybridized with the His antibody. (c) Pf measurements. In a to c, data shown are mean values ± SDs of results from 6 independent experiments. Different letters on graphs indicate significant differences based on ANOVA and Duncan's multiple new multiple range test of the data (P = .038–1.2 × 10⁻¹⁷)

FIGURE 6

De novo expression of OsPIP‐YFP with effects on Pf and drought‐tolerant responses in tobacco. (a) Subcellular localization of YFP and OsPIP‐YFP fusion proteins visualized by laser scanning confocal microscopy (LSCM) on leaves of tobacco plants 48 h after transformation with the corresponding genes. Each image represents 18 leaves from nine plants. (b) Pf measurements under the annotated conditions. (c, d) Concentrations of proline and polyamines in protoplasts incubated under the indicated conditions. In b to d, data shown are mean values ± SDs of results from six independent experiments. Different letters on graphs indicate significant differences based on ANOVA and Duncan's multiple new multiple range test of the data (P = 1.235–1.6 × 10⁻⁹)

FIGURE 7

Morphological changes of tobacco protoplasts in response to PEG₆₀₀₀ treatment. (a) Microscopic images of the protoplasts. (b) Quantification of protoplast diameters. (c) Microscopic images showing collapse of partial protoplasts. (d) Quantification of malformed protoplasts. In b and d, each image represents about 3,000 protoplasts observed in 3 independent experiments. In c and e, data shown are mean values ± SDs of results from 3 independent experiments. Asterisks indicate significant differences between the pairs of data based on ANOVA and Fisher's test (P = .005–1.6 × 10⁻⁹)

FIGURE 8

The effects of OsPIP2;2 silencing and overexpression on physical and physiological responses to PEG₆₀₀₀ treatment in rice protoplasts. (a) The protoplast Pf measurements. (b) Quantification of the protoplast decomposition. (c) and (d) Concentrations of proline and polyamines in the protoplasts. In a to d, data shown are mean values ± SDs of results from six independent experiments. Different letters on graphs indicate significant differences based on ANOVA and Duncan's multiple new multiple range test of the data (P = 1.8 × 10⁻⁷–1.2 × 10⁻¹⁶)

FIGURE 9

The effects of OsPIP2;2 silencing and overexpression on cell membrane integrity of 15‐day‐old rice seedlings growing in the absence (−) and presence (+) of PEG₆₀₀₀ treatment. (a) Electrolyte leakage from leaf segments of the plants 3 h after PEG₆₀₀₀ treatment or remained free from PEG₆₀₀₀. Data shown are mean values ± SDs of results from six independent experiments. Different letters on graphs indicate significant differences based on ANOVA and Duncan's multiple new multiple range test of the data (P = .875–1.5 × 10⁻¹⁷). (b) Rice cell membrane integrity given as reciprocals of electrolyte leakage calculated as averages based on data from a

FIGURE 10

The effects of OsPIP2;2 silencing and overexpression on expression of response genes regarded as molecular makers of the drought tolerance pathway. Rice seedlings already growing for 15 days in pot soil in a plant growth chamber were shifted into tubes containing deionized water. After 12‐h acclimation, these plants were transferred into new tubes containing deionized water only or an aqueous solution of 30% PEG₆₀₀₀. Three hours later, gene expression was analyzed by QRT‐PCR performed with total RNAs isolated from the aerial parts of the plants. The constitutively expressed OsEF1α gene was used as a reference to quantify relative expression level of an OsPIP. Data show are mean values ± SD estimates. Different letters on graphs indicate significant differences by analysis of variance (ANOVA) and Duncan's multiple new multiple range test of the data (P = .0001–1.7 × 10⁻¹¹; n = 6 independent experiments each involving 15 plants tested in three biological repeats)

FIGURE 11

Model of OsPIP2;2 functions in H₂O transport and drought tolerance. When plants are growing regularly without drought stress, OsPIP2;2 functions to facilitate H₂O transport in and out of the plant cells in response to a hydraulic gradient generated by natural metabolism in the apoplastic or cytoplasmic space. This function is assumed to play a role in maintenance of water relations. When rice plants incur osmotic stress from environment, such as the physiological drought stress caused by externally applied PEG, OsPIP2;2 turns to function to support drought tolerance possibly by physical and physiological regulatory mechanisms. In the assumed physical mechanism, the drought stress is inevitable to injure the cell membranes, causing electrolyte leakage for example, while the presence of OsPIP2;2 serves as an encountering force to help preserve the membrane integrity. The physiological mechanism, including increases in proline and polyamine concentrations, is used by OsPIP2;2 to maintain the cellular water homeostasis. Water homeostasis may also come from the role of OsPIP2;2 in modulating H₂O transportation shuttles in and out of the cells, instead of a single direction, depending on hydraulic gradient changes by electrolyte leakage. Both physical and physiological mechanisms could be integrated to increase drought tolerance intensity. Abscisic acid (ABA) signaling may partake in the regulation of OsPIP2;2‐mediated drought tolerance, which involves the ABA‐responsive transcription factor COR413‐TM1. The OsPIP2;2‐dependent tolerance pathway is also likely to have a crosstalk with signaling by H₂O₂ if it is induced by a particular stimulus (plant infection by a plant pathogen, for example) to accumulate in plant apoplastic spaces and to be transported by OsPIP2;2 to enter the cytoplasm