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Ice nucleation activity in various tissues of Rhododendron flower buds: their relevance to extraorgan freezing.

Ishikawa M, Ishikawa M, Toyomasu T, Aoki T, Price WS - Front Plant Sci (2015)

Bottom Line: The results support the following hypothesis: the high INA in bud scales functions as the subfreezing sensor, ensuring the primary freezing in bud scales at warmer subzero temperatures, which likely allows the migration of floret water to the bud scales and accumulation of icicles within the bud scales.Anti-nucleation activity (ANA) was implicated in the leachate of autoclaved bud scales, which suppresses the INA at millimolar levels of concentration and likely differs from the colligative effects of the solutes.The tissue INA levels likely contribute to the establishment of freezing behaviors by ensuring the order of freezing in the tissues: from the primary freeze to the last tissue remaining unfrozen.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences Tsukuba, Japan.

ABSTRACT
Wintering flower buds of cold hardy Rhododendron japonicum cooled slowly to subfreezing temperatures are known to undergo extraorgan freezing, whose mechanisms remain obscure. We revisited this material to demonstrate why bud scales freeze first in spite of their lower water content, why florets remain deeply supercooled and how seasonal adaptive responses occur in regard to extraorgan freezing in flower buds. We determined ice nucleation activity (INA) of various flower bud tissues using a test tube-based assay. Irrespective of collection sites, outer and inner bud scales that function as ice sinks in extraorgan freezing had high INA levels whilst florets that remain supercooled and act as a water source lacked INA. The INA level of bud scales was not high in late August when flower bud formation was ending, but increased to reach the highest level in late October just before the first autumnal freeze. The results support the following hypothesis: the high INA in bud scales functions as the subfreezing sensor, ensuring the primary freezing in bud scales at warmer subzero temperatures, which likely allows the migration of floret water to the bud scales and accumulation of icicles within the bud scales. The low INA in the florets helps them remain unfrozen by deep supercooling. The INA in the bud scales was resistant to grinding and autoclaving at 121(∘)C for 15 min, implying the intrinsic nature of the INA rather than of microbial origin, whilst the INA in stem bark was autoclaving-labile. Anti-nucleation activity (ANA) was implicated in the leachate of autoclaved bud scales, which suppresses the INA at millimolar levels of concentration and likely differs from the colligative effects of the solutes. The tissue INA levels likely contribute to the establishment of freezing behaviors by ensuring the order of freezing in the tissues: from the primary freeze to the last tissue remaining unfrozen.

No MeSH data available.


A typical result of test tube-based INA assay (A) of outer scales (OS), inner scales (IS) and florets (FL) excised at the positions shown in (B) from wintering R. japonicum flower buds collected in early December. Tubes containing a single outer scale, inner scale or floret of R. japonicum in 2 mL autoclaved Milli-Q water were cooled in 1°C decrements (1°C in 25 min) to -20°C. DW, autoclaved Milli-Q water without the specimen.
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Figure 4: A typical result of test tube-based INA assay (A) of outer scales (OS), inner scales (IS) and florets (FL) excised at the positions shown in (B) from wintering R. japonicum flower buds collected in early December. Tubes containing a single outer scale, inner scale or floret of R. japonicum in 2 mL autoclaved Milli-Q water were cooled in 1°C decrements (1°C in 25 min) to -20°C. DW, autoclaved Milli-Q water without the specimen.

Mentions: Since ice nucleation events involve probability, the number of tubes used in an INA assay is important to obtain valid data. To make a 1°C INA difference statistically significant, at least 40–50 tubes are required. The results are reported as the cumulative number of frozen tubes vs. temperature (e.g., Figure 4). To represent the INA of a sample, the median ice nucleation temperature (INT), at which 50% of the tubes froze is used rather than the mean INT, since the latter is prone to be affected by any erroneously high or low INT value. To indicate the variance of INT, ±SD is tabulated in Tables 1 and 2, which statistically means that two-thirds of the INT values are within 1 × SD range. If the sample contains numerous good ice nucleators, the median INT is shifted to warm temperatures with smaller SD. If the sample contains low amount of ice nucleators, the median INT is shifted to lower temperatures with larger SD. This relationship can be estimated from the INA determination of serially diluted ice nucleating bacteria solutions using the same 2 mL test tube assay system (data not shown). Occasionally, one might get the INT values at warm temperatures with large SD. This likely arises from the uneven occurrence of ice nucleators in the samples or from the large variance in the sample mass in the tubes.


Ice nucleation activity in various tissues of Rhododendron flower buds: their relevance to extraorgan freezing.

Ishikawa M, Ishikawa M, Toyomasu T, Aoki T, Price WS - Front Plant Sci (2015)

A typical result of test tube-based INA assay (A) of outer scales (OS), inner scales (IS) and florets (FL) excised at the positions shown in (B) from wintering R. japonicum flower buds collected in early December. Tubes containing a single outer scale, inner scale or floret of R. japonicum in 2 mL autoclaved Milli-Q water were cooled in 1°C decrements (1°C in 25 min) to -20°C. DW, autoclaved Milli-Q water without the specimen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4373250&req=5

Figure 4: A typical result of test tube-based INA assay (A) of outer scales (OS), inner scales (IS) and florets (FL) excised at the positions shown in (B) from wintering R. japonicum flower buds collected in early December. Tubes containing a single outer scale, inner scale or floret of R. japonicum in 2 mL autoclaved Milli-Q water were cooled in 1°C decrements (1°C in 25 min) to -20°C. DW, autoclaved Milli-Q water without the specimen.
Mentions: Since ice nucleation events involve probability, the number of tubes used in an INA assay is important to obtain valid data. To make a 1°C INA difference statistically significant, at least 40–50 tubes are required. The results are reported as the cumulative number of frozen tubes vs. temperature (e.g., Figure 4). To represent the INA of a sample, the median ice nucleation temperature (INT), at which 50% of the tubes froze is used rather than the mean INT, since the latter is prone to be affected by any erroneously high or low INT value. To indicate the variance of INT, ±SD is tabulated in Tables 1 and 2, which statistically means that two-thirds of the INT values are within 1 × SD range. If the sample contains numerous good ice nucleators, the median INT is shifted to warm temperatures with smaller SD. If the sample contains low amount of ice nucleators, the median INT is shifted to lower temperatures with larger SD. This relationship can be estimated from the INA determination of serially diluted ice nucleating bacteria solutions using the same 2 mL test tube assay system (data not shown). Occasionally, one might get the INT values at warm temperatures with large SD. This likely arises from the uneven occurrence of ice nucleators in the samples or from the large variance in the sample mass in the tubes.

Bottom Line: The results support the following hypothesis: the high INA in bud scales functions as the subfreezing sensor, ensuring the primary freezing in bud scales at warmer subzero temperatures, which likely allows the migration of floret water to the bud scales and accumulation of icicles within the bud scales.Anti-nucleation activity (ANA) was implicated in the leachate of autoclaved bud scales, which suppresses the INA at millimolar levels of concentration and likely differs from the colligative effects of the solutes.The tissue INA levels likely contribute to the establishment of freezing behaviors by ensuring the order of freezing in the tissues: from the primary freeze to the last tissue remaining unfrozen.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences Tsukuba, Japan.

ABSTRACT
Wintering flower buds of cold hardy Rhododendron japonicum cooled slowly to subfreezing temperatures are known to undergo extraorgan freezing, whose mechanisms remain obscure. We revisited this material to demonstrate why bud scales freeze first in spite of their lower water content, why florets remain deeply supercooled and how seasonal adaptive responses occur in regard to extraorgan freezing in flower buds. We determined ice nucleation activity (INA) of various flower bud tissues using a test tube-based assay. Irrespective of collection sites, outer and inner bud scales that function as ice sinks in extraorgan freezing had high INA levels whilst florets that remain supercooled and act as a water source lacked INA. The INA level of bud scales was not high in late August when flower bud formation was ending, but increased to reach the highest level in late October just before the first autumnal freeze. The results support the following hypothesis: the high INA in bud scales functions as the subfreezing sensor, ensuring the primary freezing in bud scales at warmer subzero temperatures, which likely allows the migration of floret water to the bud scales and accumulation of icicles within the bud scales. The low INA in the florets helps them remain unfrozen by deep supercooling. The INA in the bud scales was resistant to grinding and autoclaving at 121(∘)C for 15 min, implying the intrinsic nature of the INA rather than of microbial origin, whilst the INA in stem bark was autoclaving-labile. Anti-nucleation activity (ANA) was implicated in the leachate of autoclaved bud scales, which suppresses the INA at millimolar levels of concentration and likely differs from the colligative effects of the solutes. The tissue INA levels likely contribute to the establishment of freezing behaviors by ensuring the order of freezing in the tissues: from the primary freeze to the last tissue remaining unfrozen.

No MeSH data available.