Evaluation of Polyamine and Proline Levels during Low Temperature Acclimation of Citrus

The polyamines (PA) putrescine (Put), spermidine (Spd), and spermine (Spm) were measured during 3 weeks exposure to cold hardening (15.6°C day and 4.4°C night) and nonhardening (32.2°C day and 21.1°C night) temperature regimes in three citrus cultivars: sour orange (SO) (Citrus aurantium L.), `valencia' (VAL) (Citrus sinensis L. Osbeck), and rough lemon (RL) (Citrus jambhiri Lush). The changes in PA were compared to the amount of free proline, percent wood kill and percent leaf kill. A 2- to 3-fold increase in Spd concentrations were observed in hardened RL, SO, and VAL leaves compared to nonhardened leaves. Spermidine reached its highest level of approximately 200 nanomoles per gram fresh weight after 1 week of acclimation in both SO and VAL leaves, while RL spermidine content continued to increase up to the third week of acclimation. Spm levels in acclimated VAL and RL leaves increased 1- to 4-fold. However, SO leaves Spm content decreased with acclimation. Putrescine levels in SO and VAL increased 20 to 60% during the first 2 weeks of acclimation then declined after 3 weeks. RL putrescine content was not affected by cold acclimation. The data presented here provided direct relationship between increased Spd concentration and citrus cold hardiness. Free proline was 3- to 6-fold higher in acclimated than in nonacclimated trees. Results also demonstrate that in acclimated versus nonacclimated citrus trees the absolute amount rather than the ratio of increase in free proline is more important in predicting their ability to survive freezing stress.     

Translocation of carbohydrates and proline in young grapefruit trees at low temperatures

Girdling and defoliation of fruit-bearing grapefruit (Citrus paradisi Macf.) branches inhibited the accumulation of soluble carbohydrates and proline in fruit tissues during low temperature treatment of trees. These treatments did not inhibit hydrolysis of sucrose to reducing sugars. Flavedo and albedo tissues responded similarly to low temperatures but little or no change occurred in the juice. Therefore, soluble carbohydrates and proline do not appear to interchange between different tissues of the fruit at low temperatures but instead are translocated into the fruit from other parts of the plant. Girdling fruit-bearing branches immediately after low temperature treatments inhibited the accumulation of sucrose in fruit tissues at dehardening temperatures. Also, proline levels decreased rapidly in fruit on girdled branches at dehardening temperatures. This rapid decrease suggests proline may serve as a source for respiratory energy in grapefruit during rapidly changing temperatures that favor active growth and during recovery of citrus from environmental stress.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Changes in citrus leaf flavonoid concentrations resulting from blight-induced zinc-deficiency

Increased flavonoid concentrations were found to correlate with the elevated levels of leaf phenolic compounds occurring in blight-induced zinc-deficient citrus. In orange (Citrus sinensis L.) leaves, the increases occurred primarily in hesperidin and diosmin, whereas in grapefruit (C. paradisi Macf.) the largest increases occurred in naringin and rhoifolin. Zinc-deficiency occurring in the blighted citrus leaves appeared to be the important contributing factor to the increased flavonoid content. Although the leaves from trees with blight were typically smaller than leaves from unaffected trees, the increased flavonoid content was not significantly due to a concentration effect. Large differences occurred in the percent increases in concentrations of certain citrus leaf flavonoids. While large increases occurred for a number of flavanone and flavone glycosides, much smaller percent increases occurred for other minor flavone glycosides, and the polymethoxyflavone aglycones. The parallel increases occurring in the concentrations of certain flavone glycosides and their flavanone analogs provide a further indication that flavanone glycosides are precursors in the biosynthesis of flavone glycosides in citrus.     

Nitrogen accumulation and mobilization in Citrus leaves throughout the annual cycle

The changes of protein and amino acid contents of young and old leaves from field citrus trees [ Citrus sinensis (L.) Osbeck cv. Washington Navel] were studied throughout the year. the total protein content of old leaves decreased during the spring and summer growths, whereas in young developing leaves it increased rapidly. The end of the spring and summer flushes was followed by a quick recovery of the initial protein content due to a process of reversible senescence. The evolution of SDS-PAGE proteinograms indicated that most of the foliar proteins contributed to the nitrogen mobilization during the spring and summer growth periods. A protein band of molecular weight ca 55,000, probably containing the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, decreased relatively more than the others. The amino acid content of the leaves increased during winter time due to the accumulation of proline, which was mobilized during the spring flush. The possible nitrogen storage function of proline is discussed.     

Low temperature induction of invertase activity in grapefruit flavedo tissue

Invertase activity increased in the flavedo tissue of ‘Marsh’ grapefruit (Citrus paradisi Macf.) when trees were exposed to cold hardening temperatures and decreased at dehardening temperatures. Invertase activity also increased in the flavedo of detached fruit stored at 5δ. Reducing sugar levels paralleled invertase activity while sucrose levels were inversely related to invertase levels. The mechanism by which low temperatures induce invertase activity in grapefruit flavedo tissue was not determined. However, results indicated that a proteinaceous inhibitor, similar to the one found in potato tubers, is not involved in the regulation of invertase activity in flavedo tissue of grapefruit.     

Biochemical Alterations in Foliar Tissues of Citrus Genotypes Screened In vitro for Salinity Tolerance

Soil salinity is a prime impediment in the commercial production of citrus. In the present study two citrus rootstock genotypes viz. Citrus jambhiri and Citrus karna were cultured in vitro and exposed to NaCl salt stress. The previously standardized protocol was used for culture establishment and in vitro shoot and root regeneration. NaCl in different concentrations (25, 50, 75, 100 and 125 mM) was added in standardized regeneration and rooting media to note the biochemical changes due to salinity stress. Results revealed that salinity stress adversely affected the shoot and root differentiation and proved lethal above 100 mM NaCl. The hardening was also hampered due to salt stress. Among different biochemical parameters, proline, total soluble proteins and total sugars accumulation were enhanced however; total chlorophyll content was reduced under salinity stress. The revelation of some new protein polypeptides (21, 26 and 54 kDa) at different increasing salinity levels was attributed to their significance in stress alleviation.     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

Girdling induces oxidative damage and triggers enzymatic and non-enzymatic antioxidative defences in Citrus leaves

The effects of girdling on oxidative damage, antioxidant enzyme activity, antioxidant metabolites and proline (Pro) were studied in leaves arising from different shoot types of potted 2-year-old ‘Loretina’ mandarin (Citrus reticulata Blanco) trees during the spring flush period. Girdling increased malonyldialdehyde (MDA) and basal chlorophyll (Chl) a fluorescence (F_o) in young leaves 30 days after girdling but not in the mature leaves (ML) suggesting a disruption of photosynthetic apparatus and oxidative damage in young leaves. This phenomenon was accompanied by increasing levels of Pro. Paralleling these changes, an increase of all antioxidant enzyme activities occurred in leaves from vegetative (VG) and multiflowered leafy shoots (MLY) of girdled trees. Similarly, in ML of girdled trees, ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) activity also increased. However, dehydroascorbate reductase (DHAR) activity decreased and superoxide dismutase (SOD) activity remained unchanged. Total leaf carbohydrate content and starch also increased as a result of girdling in all shoot types. Whilst soluble sugars increased markedly in young leaves, they increased only slightly in ML. In conclusion, this study provides evidence that girdling gives rise to oxidative damage in Citrus during carbohydrate accumulation, triggering enzymatic and non-enzymatic defence mechanisms.     

Girdling induces oxidative damage and triggers enzymatic and non-enzymatic antioxidative defences in Citrus leaves

The effects of girdling on oxidative damage, antioxidant enzyme activity, antioxidant metabolites and proline (Pro) were studied in leaves arising from different shoot types of potted 2-year-old ‘Loretina’ mandarin (Citrus reticulata Blanco) trees during the spring flush period. Girdling increased malonyldialdehyde (MDA) and basal chlorophyll (Chl) a fluorescence (F_o) in young leaves 30 days after girdling but not in the mature leaves (ML) suggesting a disruption of photosynthetic apparatus and oxidative damage in young leaves. This phenomenon was accompanied by increasing levels of Pro. Paralleling these changes, an increase of all antioxidant enzyme activities occurred in leaves from vegetative (VG) and multiflowered leafy shoots (MLY) of girdled trees. Similarly, in ML of girdled trees, ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) activity also increased. However, dehydroascorbate reductase (DHAR) activity decreased and superoxide dismutase (SOD) activity remained unchanged. Total leaf carbohydrate content and starch also increased as a result of girdling in all shoot types. Whilst soluble sugars increased markedly in young leaves, they increased only slightly in ML. In conclusion, this study provides evidence that girdling gives rise to oxidative damage in Citrus during carbohydrate accumulation, triggering enzymatic and non-enzymatic defence mechanisms.     

Cold hardening in citrus stems

Stem cold hardening developed to different levels in citrus types tested in controlled environments. Exotherms indicated ice spread was more uniform and rapid in unhardened than in cold-hardened stems. All attempts to inhibit the functioning of citrus leaves resulted in less cold hardening in the stems. Citrus leaves contribute a major portion of cold hardening in the wood.     

Relationships of leaf Fatty acids to cold hardening of citrus seedlings

Three cultivars of citrus with different sensitivities to freezing temperatures (citron, Citrus medica L.; rough lemon, C. limon Burm. F; sour orange, C. aurantium L.) were cold hardened for 4 weeks. Lipids from leaves of hardened and control seedlings were fractionated and analyzed for fatty acids. The absolute amount of triglycerides and phospholipids increased in the leaves upon hardening. With hardening, total linoleic acid also increased 141% in citron, 210% in rough lemon, and 233% in sour orange. Specific increases in linoleic acid were found in triglycerides, in the four phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol), and in neutral lipids more polar than triglycerides. Trans-3-hexadecenoic acid was found only in phosphatidylglycerol.     

Involvement of cell-wall invertase in low-temperature hardening of tobacco plants

Specific features of low-temperature hardening (6 days at 8°C) of cold-sensitive tobacco plants (Nicotiana tabacum, cv. Samsun) related to changes in the cell-wall invertase activity were studied. During cold hardening, oppositely directed changes in this enzyme activity occurred in tobacco leaves and roots. In the leaves, cell-wall invertase was activated (approximately by 30%), the content of sugars increased (approximately by 25%), and the content of sucrose, the main transport form of sugars, in the apoplast reduced by three times; all these changes indicate that assimilate outflow from leaves to roots was inhibited. In contrast, in the root system, enzyme activity was decreased almost twice and the content of sugars in them was essentially unchanged. It is suggested that a strategy of low-temperature adaptation of cold-sensitive tobacco plants aimed at creating the high cold tolerance of aboveground parts, even at the expense of the root system, which, under conditions of native vegetation, is not practically exposed to damaging low temperatures.     

Solute Accumulation and Compartmentation during the Cold Acclimation of Puma Rye

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.     

Characterization of abscisic Acid-induced ethylene production in citrus leaf and tomato fruit tissues

Abscisic acid (ABA) significantly stimulated ethylene production in citrus (Citrus sinensis [L.] Osbeck, cv Shamouti orange) leaf discs. The extent of stimulation was dependent upon the concentration of ABA (0.1-1 milimolar) and the duration of treatment (15-300 minutes). Aging the discs before applying ABA increased ABA-induced ethylene production due to enhancement of both ethylene-forming enzyme activity and the responsiveness of ABA. Discs excised from mature leaves were much more responsive to ABA than discs excised from young or senescing leaves. ABA stimulated ethylene production shortly after application, suggesting that ABA does not enhance ethylene production via the acceleration of senescence. The stimulating effect of ABA on ethylene production resulted mainly from the enhancement of 1-aminocylopropane-1-carboxylic acid synthesis. Stimulation of ethylene production by ABA in intact citrus leaves and tomato (Lycopersicon esculentum Mill., cv Castlemart) fruit was small but could be increased by various forms of wounding.     

De novo arginine biosynthesis in leaves of phosphorus-deficient citrus and poncirus species

Young, fully expanded leaves from 7-month-old P-deficient citrus rootstock seedlings had levels of nonprotein arginine that were 10- to 50-fold greater than those from P-sufficient control plants. Arginine content of the protein fraction increased 2- to 4-fold in P-deficient leaves. Total arginine content, which averaged 72 ± 6 micromoles per gram dry weight of P-sufficient leaf tissue (mean ± se, n = the four rootstocks) was 207, 308, 241, and 178 micromoles in P-deficient leaves from Citrus limon cv rough lemon, Poncirus trifoliata × C. sinensis cv Carrizo citrange and cv Troyer citrange, and P. trifoliata cv Australian trifoliate orange, respectively. For each rootstock, the accumulation of arginine paralleled an increase in the activity of the pathway for the de novo biosynthesis of arginine. The ratio of the nanomoles NaH¹⁴CO₃ incorporated into the combined pool of arginine plus urea per gram fresh weight intact leaf tissue during a 3-hour labeling period for P-deficient to P-sufficient plants was 91:34, 49:11, 35:11, and 52:41, respectively. When P-deficient plants were supplied with P, incorporation of NaH¹⁴CO₃ into arginine plus urea was reduced to the level observed for the P-sufficient control plants of the same age and arginine ceased to accumulate. Arginase and arginine decarboxylase activity were either unaffected or slightly increased during phosphorus deficiency. Taken together, these results provide strong evidence that arginine accumulation during phosphorus deficiency is due to increased activity of the de novo arginine biosynthetic pathway.     

Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro

The use of in vitro shoot cultures to evaluate osmotic and salt tolerance and the effects of salt and mannitol in the medium on proline and sugar accumulation were investigated in two poplar species, P. euphratica and P. alba cv. Pyramidalis × P. tomentosa. Shoot length, leaf number, whole plant dry weight, and the accumulation of proline and total soluble sugars in leaves were quantified after 2 weeks. All P. euphratica plantlets survived at all levels of mannitol and NaCl, while the mortality of P. alba cv. Pyramidalis × P. tomentosa increased both at the mannitol and the NaCl treatments. A significant increase in proline accumulation was observed in both young and mature P. euphratica leaves at 200 mM mannitol and above, and at 150 mM NaCl and above. The total soluble sugar content increased in young P. euphratica leaves at 250 mM NaCl; however, it decreased in the mature leaves. Similar increases of the total soluble sugar content were not seen in P. alba cv. Pyramidalis × P. tomentosa plants in response to either mannitol or NaCl treatment. Our results suggest that accumulated proline and sugars promote osmotic and salt tolerance. The effects of accumulated proline and total soluble sugars on leaves are discussed in relation to growth and osmotic adjustment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sugar regulation of plastid interconversions in epicarp of citrus fruit

Seasonal transformations between chloroplasts and chromoplasts, as measured by changes in chlorophyll content, in the epicarp of degreening and regreening Citrus sinensis (L.) Osbeck cv Valencia fruit closely parallelled the accumulation and later loss of soluble sugars. At any stage of development, reversing the relative soluble sugar content in the epicarp by culturing pericarp segments on agar media with low (15 millimolar) or high (150 millimolar) sucrose concentrations reversed the direction of change in chlorophyll content. Fruit of C. madurensis Lour., which mature year around and do not regreen, also accumulated soluble sugars in the pericarp as degreening was initiated.

The epicarp of C. sinensis fruit accumulated nitrogen, but total nitrogen concentrations and amino acid concentrations changed little, during degreening and regreening of C. sinensis fruit. Cessation of nitrogen fertilization reduced the tendency of pericarp segments to regreen in vitro during subsequent years, but regreening tendency was restored by inclusion of KNO₃ in the media.

It is concluded that chloroplasts become chromoplasts and citrus fruit degreen partially in response to the accumulation of sugars in the epicarp and that the reverse transformation accompanying regreening of certain citrus species occurs when accumulated sugars disappear. Change in nitrogen flux to the fruit is probably not a factor in regulating seasonal transformations, but an abundance of nitrogen in the epicarp diminishes the effects of high sugar concentrations in inducing transformation of chloroplasts to chromoplasts, thereby retarding degreening and promoting regreening.