Aleast two types of low-temperature injury are known to occur in plant

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1 [271 ] A FORM OF LOW-TEMPERATURE INJURY. IN DETACHED LEAVES BY E. R. ROUX University of Cape Town (With 2 figures in the text) Aleast two types of low-temperature injury are known to occur in plant tissues. At temperatures below the freezing-point of the cell sap changes occur which result in the death of the cells. There is a considerable literature on the mechanism of frost resistance in plants, and injury due to freezing has been extensively investigated. On the other hand, at temperatures above the freezingpoint of the cell sap, various forms of low-temperature injury or "physiological breakdown" have been described. These changes do not necessarily cause the immediate death of the cells, though metabolism may be obviously affected in a number of ways. Examples of forms of low-temperature injury belonging to this class have hitherto heen recorded only fot fruits. They include browning of the tissues in plums, "woolliness" in peaches, and the pitting of the skin in grapefruit. The "sweetening" of potatoes at low temperatures (Barker, 1933) may be a parallel phenomenon. The kinetic aspects of cold injury in fruit have been examined by van der Plank & Davies (1937), who found in many cases temperatures of maximum injury, with "safe" regions above and below the critical region. It is recognized that fruit exhibits considerable variation, individual and seasonal, in its susceptibility to low-temperature injury. It is generally held that unripe fruit is more susceptible than ripe fruit. Davies and his co-workers (1936) have shown that peaches may be protected against the development of woolliness by keeping the fruit for a few days at a high temperature before cooling. Peaches left on the tree till thoroughly ripe never develop woolliness in store. Donen (1939) has shown that there is an inverse relation between sorbitol content and lowtemperature browning in the Kelsey plum. As far as the writer is aware, low-temperature physiological breakdown has hitherto not been described in foliage leaves. In the course of experiments on the respiration of detached leaves of the loquat {Eriobotrya japonica), kept in the dark with their petioles in water, it was found that leaves picked in the winter and kept at temperatures in the neighbourhood of 2 C. became brown in patches when the temperature was subsequently raised. The browning was found to begin at the periphery of the leaf and between the main veins. It was accompanied by a sudden rise in CO2 output followed by a fall. The cells in the affected areas eventually died.

2 272 E. R. Roux Leaves kept at 25 C. for a few days before being placed in cold store developed a high degree of immunity to low-temperature injury. Low-temperature browning in leaves could therefore be controlled in exactly the same way as woolliness iri peaches. Attempts were made to discover whether similar phenomena could be demonstrated in leaves from other plants. It was found in most of tbe species investigated that the leaves would not stand up to tbe experimental treatment but wilted after a day or two. Of the leaves which did not wilt two evergreen types Viburnum sp. and Eicus sp. showed physiological breakdown after exposure to 2 C. None of the leaves from herbaceous plants showed any visible injury after storage at tbis temperature. These included Tropaeolum mafus, Hilianthus annuus, Statice sp. and Senecio sp. EXPERIMENTAL For each experiment a number of leaves, as nearly uniform in age and appearance as possible, were picked from a single tree. Tbey were placed witb their petioles in water and taken to tbe laboratory as quickly as possible. Unless otherwise stated, fully mature leaves over six months old were used. They were divided into a number of sets, usually 4-6 leaves per set. During cold storage they were kept in open containers. For pre-storage or post-storage at 25 C. tbey were placed in closed chambers ventilated by an air current and immersed in a constant temperature bath. Leaves were kept in darkness witb their petioles in water throughout. The amount of browning, expressed as a percentage of the total leaf area, was measured by placing the leaves between sbeets of glass, illuminating them from below, and tracing their outlines and the configuration of the brown and green areas on sheets of paper. Tbe areas were then calculated by cutting out and weighing tbe paper. Development of browning zvith time Curves showing the development of browning witb time are given in Fig. i for loquat leaves picked in 1937 and The temperature used was 2 C. and the leaves were placed in cold store immediately after picking. Sets were taken out at intervals and post-stored at 25 C. for 24 hr., when tbe amount of browning was measured. It will be seen that a minimum period at the low temperature is required to induce physiological injury. Leaves exposed to 2 C. for less tban tbis period showed no browning when transferred to 25 C. and kept there for 7 days. Tbe length of the induction period was much shorter in 1937 tban in 1939, but the browning wben it did develop was more severe in In general browning only developed after removal from cold store, spreading rapidly for the first 24 br. and then very slowly. Leaves kept at 2 C. for 20 days already showed a certain amount of browning before removal. Browning at different temperatures Sets of loquat leaves from a single picking were placed immediately at 5, o, 2 and 5 C, and kept at tbese temperatures for 10 days. They were then transferred to 25 C. and tbe amount of browning measured after 24 br. Leaves kept at 5 C.

3 A form of low-temperature injury in detached leaves were killed by the treatment. Browning after exposure to o, 2 and 5 C. was 57, 42 and o % respectively. Effects of pre-storage at high temperatures A number of experiments were done with leaves of loquat. Viburnum and wild fig to determine the effects of high temperature pre-storage on low-temperature browning following exposure to 2 C. Results obtained with loquat leaves in 1937 and 1939 are shown in Fig. 2. In 1937 the leaves were kept at 2 C. for 7 days, in 1939 for 10 days. Pre-storage in both cases was at 25 C. The protective effect of pre-storage was clearly demonstrated Days' storage at 2 C. Fig. I. Progress of browning with time in loquat leaves stored at 2 C. A, leaves picked 13 August B, leaves picked 9 June A similar result was obtained with leaves of Viburnum cold-stored at 2 C. for 30 days (see Fig. 2). In this case laboratory temperature (about 18 C.) was used for pre-storage. An appreciable amount of injury, in the form of darkened areas, became visible during cold storage. Consequently the leaves were not post-stored, the amount of injury being measured immediately after removal from cold store. Leaves of a wild fig (Ficus sp.) picked on 23 June 1939 were stored at 2 C. for 7 days. A control set was kept at laboratory temperature for 7 days and then placed for 7 days at 2 C. Three days after removal from cold store the first set showed New Phytol. 39,

4 274 E. R. Roux injury equal to 36% of the total area. The control failed to show any injury even when kept for 8 days after removal from store. Seven days' pre-storage at laboratory temperature therefore conferred complete immunity on these leaves. The relative susceptibility of mature and immature leaves of the loquat was determined in another experiment. Immature leaves developed 91% browning after 7 days at 2 C. followed by 24 hr. at room temperature. Controls kept at room 10 Days' pre-storage 15 Fig. 2. Effect of pre-storage on amount of browning developed by leaves stored at 2 C. A, loquat leaves picked 13 August 1937, kept at 2 C. for 7 days. B, loquat leaves picked 9 June 1939 kept at 2" C. for 10 days. C, Viburnum leaves, kept at 2 C. for 30 days. A and B pre-stored and poststored at 25 C. C pre-stored at laboratory temperature (about 18 C). temperature for 7 days, then placed at 2 C. for 7 days, then transferred back to room temperature for 24 hr., gave no browning. Mature leaves, picked from the same tree at the same time and treated in the same way, gave 20 % browning as against less than i % for the pre-stored controls. It appeared therefore that young leaves were much more susceptible than old leaves to low-temperature injury and responded equally well (or even better) to pre-storage treatment.

5 A form of low-temperature injury in detached leaves 275 Seasonal incidence of susceptibility to low-temperature injury In the tbree types of evergreen leaf studied it was found tbat only during a limited period in the winter were tbey subject to tbe form of low-temperature injury described. Tbe actual period of susceptibility varied considerably from year to year. Browning after exposure to 2 C. was not observed during tbree seasons except in tbe months June-August. In 1937 it appeared as late as 13 August, but in 1939 it was confined to a period of about 20 days in June. In Cape Town during tbe winter atmospberic temperatures are lower (tbough frosts are practically unknown), sunshine hours less and relative humidities higher than during tbe summer, and there may be some general relation between tbese factors and susceptibility to low-temperature injury. But attempts to relate the amount of browning to tbe meteorological conditions immediately preceding tbe date of picking were not successful. Tbe phenomena are obviously very complex and numerous "external" and "internal" factors must be involved. Since immunity is acquired during starvation, it might be suggested that conditions wbicb favour assimilation prior to picking might be an important factor in producing susceptibility. On the otber band, summer conditions almost certainly lead to increased rates of assimilation, and leaves during the summer never showed low temperature physiological breakdown. An investigation of tbe carbobydrate metabolism of susceptible and non-susceptible leaves migbt tbrow some ligbt on tbe problem, but tbis bas so far not been attempted. It is interesting tbat leaves should be more susceptible to low-temperature injury in tbe winter tban in tbe summer. Meyer (1932) states tbat "evergreen leaves are not, in general, resistant to cold during tbe warmer months of tbe year". But this refers to letbal injury at. temperatures below tbe freezing point of the cell sap. Physiological injury at temperatures sufficiently low, but above the freezingpoint, evidently bas a totally different seasonal incidence. Kidd & West (1933) have shown in the case of English-grown bot-bouse tomatoes tbat "summer-grown fruit tolerates low temperature (5 C.) better tban autumn-grown fruit", and Wardlaw (1939) has pointed to tbe bigb degree of resistance to cold injury of tomatoes grown under tropical conditions in Trinidad. SUMMARY A form of low-temperature browning, or "physiological injury" following exposure to temperatures above tbe freezing-point of tbe cell sap, is described for detacbed leaves of certain evergreen trees. Leaves picked in the summer are not subject to tbis form of injury. Leaves otherwise susceptible may be protected against low-temperature injury by exposing them for a few days to a bigher temperature before they are placed in cold store. Tbis resembles tbe metbod by wbich "woolliness" in peacbes is controlled in commercial practice. Attempts to relate susceptibility to low-temperature injury in winter leaves to 18-2

6 276 E. R. Roux atmospheric temperatures, humidities and light intensities at the time of picking were not successful. The writer held a Carnegie grant during part of the time in which this work was carried out. Thanks are due to the Royal Observatory, Cape Town, and the Government Meteorological Station, Wingfield Aerodrome, for meteorological data supplied. REFERENCES BARKER, J. (1933). Analytic studies in plant respiration. IV and V. The relation of the respiration of potatoes to the concentration of sugars and to the accumulation of a depressant at low temperatures. Proc. roy, Soc. B, 112, 316. DAVIES, R., BOYES, W. W. & DE VILLIEHS, D. J. R. (1936). Cold storage of peaches. Report of the Lozo Temperature Research Laboratory, Cape Toion, p DoNEN, I. (1939). The role of sorbitol in the carbon metabolism of the Kelsey plum. Biochem. J KiDD, F. & WEST, C. (1933). Low-temperature tolerance of summer- and autumn-grown hot-house tomatoes. Rep, Fd Invest. Bd, p. 82. MEYER, B. S. (1932). Further studies on cold resistance in evergreens, with special reference to the possible role of bound water. Bot. Gaz, 94, 297. VAN DER PLANK, J. E. & DAVIES, R. (1937). Temperature cold injury curves of fruit. I. General thermochemical considerations. J. Pomol. 15, 226., C. W. (1939). Storage of tropical fruit. Nature, Lond., 144, 178.

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