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pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NOFormula.

Greenway H, Kulichikhin KY, Cawthray GR, Colmer TD - J. Exp. Bot. (2011)

Bottom Line: Net H(+) influx (μmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ∼1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports.NO(3)(-) reduction presumably functioned as a biochemical pHstat.Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.

View Article: PubMed Central - PubMed

Affiliation: The University of Western Australia, Crawley, WA, Australia.

ABSTRACT
During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H(+) influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K(+) efflux was continuous. Further experiments used excised coleoptile tips (7-10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO(3)(-), which distinguished two processes involved in pH regulation. Net H(+) influx (μmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ∼1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports. NO(3)(-) reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K(+) balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO(3)(-) supply. Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.

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Amino acids (μmol g−1 fresh weight) in excised tips of rice coleoptiles under anoxia in the presence of 0.2 mM NO3−NO3− at pH 6.5 and after 60 h at either pH 6.5 or 3.5. (A) L-alanine, (B) γ-aminobutyric acid (GABA). The start of anoxia is at 0 h. pH 6.5, open squares, thin line; pH 3.5, closed squares, bold line. Data are the means of three experiments, each with three replicates. Values at 0 h are tissues sampled at the end of an 18 h pre-treatment at 0.05 mM O2. In one experiment, coleoptile tips were returned at 78 h from pH 3.5 to 6.5 while maintaining anoxia, and after another 18 h L-alanine had decreased from 30 to 21 (μmol g−1 fresh weight); no data for γ-aminobutyric acid are available after this shift.
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fig3: Amino acids (μmol g−1 fresh weight) in excised tips of rice coleoptiles under anoxia in the presence of 0.2 mM NO3−NO3− at pH 6.5 and after 60 h at either pH 6.5 or 3.5. (A) L-alanine, (B) γ-aminobutyric acid (GABA). The start of anoxia is at 0 h. pH 6.5, open squares, thin line; pH 3.5, closed squares, bold line. Data are the means of three experiments, each with three replicates. Values at 0 h are tissues sampled at the end of an 18 h pre-treatment at 0.05 mM O2. In one experiment, coleoptile tips were returned at 78 h from pH 3.5 to 6.5 while maintaining anoxia, and after another 18 h L-alanine had decreased from 30 to 21 (μmol g−1 fresh weight); no data for γ-aminobutyric acid are available after this shift.

Mentions: We then measured endogenous organic solutes involved in a possible biochemical pHstat for coleoptile tips at pH 6.5 or 3.5 without β-alanine. During the first 60 h anoxia at pH 6.5, there were no consistent changes in malate concentration, which ranged between 9 and 10 μmol g−1 fresh weight, while there was net succinate formation of ∼2.7 μmol g−1 fresh weight (Fig. 2). Exposure to pH 3.5 during anoxia decreased the concentrations of malate from 9–10 to ∼3.8 μmol g−1 fresh weight during the first 18 h, with further decreases to ∼2 μmol g−1 fresh weight during the next 18 h at pH 3.5 (Fig. 2). Succinate also decreased at pH 3.5 although more gradually than malate (Fig. 2). Fumarate was at very low levels but followed a similar trend to malate and succinate (Fig. 2). Anoxia at pH 6.5 greatly increased the L-alanine concentration from 2 to ∼30 μmol g−1 fresh weight over the first 60 h of anoxia and then to ∼43 μmol g−1 fresh weight over the next 40 h (Fig. 3A). After transfer of coleoptile tips to pH 3.5 at 60 h anoxia, the means of three experiments showed no appreciable changes in L-alanine (Fig. 3A), while there was an appreciable increase in a fourth experiment (although this was only about half of the increase at pH 6.5; Fig. 4B). At pH 6.5, γ-aminobutyric acid increased from ∼1.5 to 2.8 μmol g−1 fresh weight during the first 60 h in anoxia; after 60 h the increase at pH 6.5 slowed but accelerated at pH 3.5, and this increase after 60 h was ∼8-fold greater at pH 3.5 than at pH 6.5 (Fig. 3B). Furthermore, the analyses showed that serine increased from 1.6 μmol g−1 fresh weight at the end of hypoxia to 3–4.6 μmol g−1 fresh weight during anoxia, with little difference between pH treatments.


pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NOFormula.

Greenway H, Kulichikhin KY, Cawthray GR, Colmer TD - J. Exp. Bot. (2011)

Amino acids (μmol g−1 fresh weight) in excised tips of rice coleoptiles under anoxia in the presence of 0.2 mM NO3−NO3− at pH 6.5 and after 60 h at either pH 6.5 or 3.5. (A) L-alanine, (B) γ-aminobutyric acid (GABA). The start of anoxia is at 0 h. pH 6.5, open squares, thin line; pH 3.5, closed squares, bold line. Data are the means of three experiments, each with three replicates. Values at 0 h are tissues sampled at the end of an 18 h pre-treatment at 0.05 mM O2. In one experiment, coleoptile tips were returned at 78 h from pH 3.5 to 6.5 while maintaining anoxia, and after another 18 h L-alanine had decreased from 30 to 21 (μmol g−1 fresh weight); no data for γ-aminobutyric acid are available after this shift.
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fig3: Amino acids (μmol g−1 fresh weight) in excised tips of rice coleoptiles under anoxia in the presence of 0.2 mM NO3−NO3− at pH 6.5 and after 60 h at either pH 6.5 or 3.5. (A) L-alanine, (B) γ-aminobutyric acid (GABA). The start of anoxia is at 0 h. pH 6.5, open squares, thin line; pH 3.5, closed squares, bold line. Data are the means of three experiments, each with three replicates. Values at 0 h are tissues sampled at the end of an 18 h pre-treatment at 0.05 mM O2. In one experiment, coleoptile tips were returned at 78 h from pH 3.5 to 6.5 while maintaining anoxia, and after another 18 h L-alanine had decreased from 30 to 21 (μmol g−1 fresh weight); no data for γ-aminobutyric acid are available after this shift.
Mentions: We then measured endogenous organic solutes involved in a possible biochemical pHstat for coleoptile tips at pH 6.5 or 3.5 without β-alanine. During the first 60 h anoxia at pH 6.5, there were no consistent changes in malate concentration, which ranged between 9 and 10 μmol g−1 fresh weight, while there was net succinate formation of ∼2.7 μmol g−1 fresh weight (Fig. 2). Exposure to pH 3.5 during anoxia decreased the concentrations of malate from 9–10 to ∼3.8 μmol g−1 fresh weight during the first 18 h, with further decreases to ∼2 μmol g−1 fresh weight during the next 18 h at pH 3.5 (Fig. 2). Succinate also decreased at pH 3.5 although more gradually than malate (Fig. 2). Fumarate was at very low levels but followed a similar trend to malate and succinate (Fig. 2). Anoxia at pH 6.5 greatly increased the L-alanine concentration from 2 to ∼30 μmol g−1 fresh weight over the first 60 h of anoxia and then to ∼43 μmol g−1 fresh weight over the next 40 h (Fig. 3A). After transfer of coleoptile tips to pH 3.5 at 60 h anoxia, the means of three experiments showed no appreciable changes in L-alanine (Fig. 3A), while there was an appreciable increase in a fourth experiment (although this was only about half of the increase at pH 6.5; Fig. 4B). At pH 6.5, γ-aminobutyric acid increased from ∼1.5 to 2.8 μmol g−1 fresh weight during the first 60 h in anoxia; after 60 h the increase at pH 6.5 slowed but accelerated at pH 3.5, and this increase after 60 h was ∼8-fold greater at pH 3.5 than at pH 6.5 (Fig. 3B). Furthermore, the analyses showed that serine increased from 1.6 μmol g−1 fresh weight at the end of hypoxia to 3–4.6 μmol g−1 fresh weight during anoxia, with little difference between pH treatments.

Bottom Line: Net H(+) influx (μmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ∼1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports.NO(3)(-) reduction presumably functioned as a biochemical pHstat.Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.

View Article: PubMed Central - PubMed

Affiliation: The University of Western Australia, Crawley, WA, Australia.

ABSTRACT
During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H(+) influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K(+) efflux was continuous. Further experiments used excised coleoptile tips (7-10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO(3)(-), which distinguished two processes involved in pH regulation. Net H(+) influx (μmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ∼1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports. NO(3)(-) reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K(+) balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO(3)(-) supply. Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.

Show MeSH
Related in: MedlinePlus