Effects of chilling temperature on photosynthetic rates, photosynthetic enzyme activities and metabolite levels in leaves of three sugarcane species

FBPase, fructose-1,6-bisphosphatase
NADP-MDH, NADP-malate dehydrogenase
NADP-ME, NADP-malic enzyme
OAA, oxaloacetic acid
PEP, phosphoenolpyruvate
PEPcase, phosphoenolpyruvate carboxylase
PPDK, pyruvate orthophosphate dikinase
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase

The aim of this study was to investigate the mechanism of photosynthetic changes in sugarcane leaves in response to chilling temperature by using three species ( Saccharum sinense R. cv. Yomitanzan, Saccharum sp. cv. NiF4 and Saccharum officinarum L. cv. Badira) differing in origin and cold sensitivity. Yomitanzan is native to subtropical areas, Badira is native to tropical areas and NiF4 is a hybrid species containing genes of both tropical and subtropical species. At exposure to chilling temperature (10 °C), the photosynthetic rate in the leaves at either 10 °C or 30 °C showed a greater decrease in Badira than in NiF4 and Yomitanzan. After 28 h exposure of plants to the chilling temperature, the extractable activities of pyruvate, orthophosphate dikinase (PPDK) and NADP-malate dehydrogenase (NADP-MDH) increased or were relatively stable in the leaves of NiF4 and Yomitanzan, but decreased substantially in Badira. Correspondingly, there was a substantial accumulation of aspartate, and the level of alanine increased in Badira leaves during the chilling treatment. It is suggested that NADP-MDH and PPDK are key enzymes which may determine the cold sensitivity in photosynthesis of sugarcane.     

Up-regulation of sucrose phosphate synthase in rice grown under elevated CO2 and temperature

Rice (Oryza sativa L. cv. IR-30) was grown season-long in outdoor, controlled-environment chambers at 33 Pa CO₂ with day/night/paddy-water temperatures of 28/21/25 °C, and at 66 Pa CO₂ with five different day/night/paddy-water temperature regimes (25/18/21, 28/21/25, 31/24/28, 34/27/31 and 37/30/34 °C). Sucrose phosphate synthase (SPS) activities in leaf extracts at 21, 48 and 81 days after planting (DAP) were assayed under saturating and selective (limiting) conditions. Diel SPS activity data indicated that rice SPS was light regulated; with up to 2.2-fold higher rates during the day. Throughout the growth season, leaf SPS activities were up-regulated in the CO₂-enriched plants, averaging 20 and 12% higher than in ambient-CO₂ grown plants in selective and saturating assays, respectively. Similarly, SPS activities increased 2.4% for each 1 °C rise in growth temperature from 25 to 34 °C, but de creased 11.5% at 37 °C. Leaf sucrose content was higher, and mirrored SPS activity better, than starch, although starch was more responsive to CO₂ treatment. Leaf sucrose and starch contents were significantly higher throughout the season in plants at elevated CO₂, but the N content averaged 6.5% lower. Increasing growth temperatures from 25 to 37 °C caused a linear decrease (62%) in leaf starch content, but not in sucrose. Consequently, the starch:sucrose ratio declined with growth temperature. The data are consistent with the hypothesis that the up-regulation of leaf SPS may be an acclimation response of rice to optimize the utilization and export of organic-C with the increased rates of inorganic-C fixation in elevated CO₂ or temperature growth regimes.     

Relations between carbohydrate accumulation in leaves, sucrose phosphate synthase activity and photoassimilate transport in chilling treated maize seedlings

Sucrose and starch concentration, sucrose phosphate synthase (SPS) activity in leaves, and long distance transport were studied in maize seedlings treated with moderate chilling (14 °C/12 °C - day/night). Two inbred lines were tested: chilling-tolerant KW1074 and chilling-sensitive CM109. Seedlings were grown in phytotrone on water nutrient until the 4-th leaf appearance. The estimations were done on fully developed 2-nd leaf. Six days after the temperature was lowered, leaves of line KW 1074 plants contained 5-fold more sucrose and starch than the control ones. The same treatment of CM 109 seedlings resulted in accumulation of sucrose and starch by 2-fold and 8.5-fold, respectively. As the result of chilling-treatment, ¹⁴C assimilation rate (P_a), transport speed in the leaf blade (TS₁) and along the plant (TS_m) decreased by about 50 % in both lines. On the other hand, time necessary for radiolabel movement into the phloem loading region (AT) increased strongly, especially in chilling-sensitive line CM 109. It was also noted, that the radioactivity exported from leaves (R₁) and imported by roots (R_m) decreased in line CM 109, and increased slightly in line KW 1074. The activity of SPS extracted from leaves of both lines decreased by about 3.3 when temperature was lowered form 30°C to 10°C. There was no effect of 6 day treatment of chilling on SPS activity. Changes in sucrose and starch concentration, SPS activity as well as differences in transport parameters observed in KW1074 and CM109 seedlings at moderate low temperatures are discussed in terms of mechanism of maize chilling-sensitivity.     

The Decreased Cold-Resistance of Chilling-Sensitive Plants Is Related to Suppressed CO2 Assimilation in Leaves and Sugar Accumulation in Roots

Tomato (Lycopersicon esculentum L., cv. Sibirskii skorospelyi) and cucumber (Cucumis sativus L., cv. Konkurent) plants were grown in a soil culture in a greenhouse at an average daily temperature of 20°C and ambient illumination until the development of five and eight true leaves, respectively. During the subsequent three days, some plants were kept in a climatic chamber at 6°C in the light, whereas other plants remained in a greenhouse (control). The cold-resistance of cucumber leaves and roots, as assayed from the electrolyte leakage, was reduced after cold exposure stronger than cold-resistance of tomato organs. The ratio photosynthesis/dark respiration was lower in cucumber than in tomato leaves at all measurement temperatures. The concentrations of sugars (sucrose + glucose + fructose) increased in chilled tomato roots but decreased in cucumber roots. Cold exposure changed the activities of various invertase forms (soluble and insoluble acidic and alkaline invertases). The total invertase activity and the ratio of mono- to disaccharides increased. The lower cucumber cold-resistance is related to the higher sensitivity of its photosynthetic apparatus to chilling and, as a consequence, insufficient root supply with sugars.     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

去果河套蜜瓜源叶碳水化合物及其相关酶昼夜变化特征

以河套蜜瓜为试材,在果实迅速膨大期通过去果处理改变库源关系,研究源叶净光合速率,蔗糖、还原糖和淀粉含量及其代谢相关酶活性的昼夜变化规律。结果表明:(1)源叶的净光合速率为单峰曲线,无明显的"光合午休"现象,去果处理对其无影响。(2)源叶中蔗糖和还原糖含量的昼夜变化为单峰曲线,蔗糖磷酸合成酶和蔗糖合成酶合成方向活性的昼夜变化为双峰曲线,蔗糖合成酶分解方向、酸性转化酶和中性转化酶活性的昼夜变化无明显规律,改变库源关系对这些指标均无显著影响;蔗糖含量升高受蔗糖磷酸合成酶和蔗糖合成酶合成方向正调控,而蔗糖含量降低则受多种酶的共同调节。(3)源叶中淀粉含量和腺苷二磷酸葡萄糖焦磷酸化酶活性的昼夜变化为单峰曲线,去果处理可以显著提高淀粉含量和腺苷二磷酸葡萄糖焦磷酸化酶活性,淀粉含量升高受腺苷二磷酸葡萄糖焦磷酸化酶正调控。     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Mulberry Leaf Metabolism under High Temperature Stress

Effects of high temperature on the activity of photosynthetic enzymes and leaf proteins were studied in mulberry (Morus alba L. cv. BC2-59). A series of experiments were conducted at regular intervals (120, 240 and 360 min) to characterize changes in activities of ribulose-1,5-bisphosphate carboxylase (RuBPC) and sucrose phosphate synthase (SPS), photosystem 2 (PS 2) activity, chlorophyll (Chl), carotenoid (Car), starch, sucrose (Suc), amino acid, free proline, protein and nucleic acid contents in leaves under high temperature (40 °C) treatments. High temperature markedly reduced the activities of RuBPC and SPS in leaf extracts. Chl content and PS 2 activity in isolated chloroplasts were also affected by high temperature, particularly over 360 min treatment. Increased leaf temperature affected sugar metabolism through reductions in leaf starch content and sucrose-starch balance. While total soluble protein content decreased under heat, total amino acid content increased. Proline accumulation (1.5-fold) was noticed in high temperature-stressed leaves. A reduction in the contents of foliar nitrogen and nucleic acids (DNA and RNA) was also noticed. SDS-PAGE protein profile showed few additional proteins (68 and 85 kDa) in mulberry plants under heat stress compared to control plants. Our results clearly suggest that mulberry plants are very sensitive to high temperature with particular reference to the photosynthetic carbon metabolism.     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Changes in antioxidant enzyme activities in detached leaves of cucumber exposed to chilling

We studied changes in biochemical and physiological status, level of oxidative damage, and antioxidant enzyme activities in detached leaves of cucumber plants (Cucumis sativus L. cv. Pyunggangnaebyungsamchuk) that were exposed to a low temperature (4°C). Chlorophyll fluorescence (F_v/F_m) declined during the chilling treatment, but was slowly restored after the tissues were returned to 25°C. Likewise, the fluorescence quenching coefficient and relative water content decreased during the stress period, but then increased during recovery. In contrast, we detected a significant rise in protein and hydrogen peroxide contents in the chilled leaves, as well as higher activities for superoxide dismutase, ascorbate peroxidase, peroxidase, and glutathione reductase. However, the level of catalase decreased not only during chilling but also after 24 h of recovery. These results indicate that exposure to low temperatures acts as an oxidative stress. Moreover, we propose that a regulating mechanism exists in the detached cucumber leaves and contains an antioxidant defense system that induces active oxygen species, thereby alleviating the effects of chilling stress within 12 h.     

Increase of sucrose synthase activity in wheat plants after a chilling shock

Plants of wheat (Triticum aestivum) were grown at 23°C. After 17 days they were suddenly transferred to 4°C under the same light conditions. The change in temperature produced an increase in the level of sucrose and fructans. Following the chilling shock, enzymes related to sucrose metabolism were measured. The activities of fructose 1,6-biphosphatase, UDPGlc pyrophosphorylase, sucrose phosphate synthase (SPS), UDPase and invertase were not modified even after 8 days at 4°C. On the contrary, the activity of sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) rose continuously, immediately after the chilling shock.     

Sucrose Metabolism at Three Leaf Development Stages in Bean Plants

Sucrose metabolism was studied at three leaf development stages in two Phaseolus vulgaris L. cultivars, Tacarigua and Montalban. The changes of enzyme activities involved in sucrose metabolism at the leaf development stages were: (1) Sink (9-11 % full leaf expansion, FLE): low total sucrose phosphate synthase (SPS) activity, and higher acid invertase (AI) activity accompanied by low sucrose synthase (SuSy) synthetic and sucrolytic activities. (2) Sink to source transition (40-47 % FLE): increase in total SPS and SuSy activities, decrease in AI activity. (3) Source (96-97 % FLE): high total SPS activity, increased SuSy activities, decreased AI activity. The hexose/sucrose ratio decreased from sink to source leaves in both bean cultivars. The neutral invertase activity was lower than that of AI; it showed an insignificant decrease during the sink-source transition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi

The rhythm of CO₂ assimilation exhibited by leaves of Bryophyllum fedtschenkoi maintained in light and normal air occurs only at constant ambient temperatures between 10°C and 30°C. Over this range the period increases linearly with increasing temperature from the extremely low value of 15.7 h to 23.3 h, but shows a considerable degree of temperature compensation. Outside the range 10°C–30°C the rhythm is inhibited but re-starts on changing the temperature to 15°C. Prolonged exposure of leaves to high (40°C) and low (2°C) temperature inhibits the rhythm by driving the basic oscillator to fixed phase points in the cycle which differ by 180°, and which have been characterised in terms of the malate status of the leaf cells. At both temperatures loss of the circadian rhythm of CO₂ assimilation is due to the inhibition of phosphoenolpyruvate carboxylase (PEPCase) activity, but the inhibition is apparently achieved in different ways at 40°C and 2°C. High temperature appears to inhibit directly PEPCase activity, but not the activity of the enzymes responsible for the breakdown of malate, with the result that the leaf acquires a low malate status. In contrast, low temperature does not directly inhibit PEPCase activity, but does inhibit enzymes responsible for malate breakdown, so that the malate level in the leaf increases to a high value and PEPCase is eventually allosterically inhibited. The different malate status of leaves held at these two temperatures accounts for the phases of the rhythms being reversed on returning the leaves to 15°C. After exposure to high temperature, CO₂ fixation by PEPCase activity can begin immediately, whereas after exposure to low temperature, the large amount of malate accumulated in the leaves has to be decarboxylated before CO₂ fixation can begin.     

Long-term chilling of young tomato plants under low light and subsequent recovery

The influence of unfavourable climatic conditions at the onset of the growth period on chilling-sensitive tomato (Lycopersicon esculentum Mill., cv. Abunda) was studied by exposing young plants to combinations of low temperature and low light (60–100 mol quanta · m^–2 · s^–1) for several weeks. When the temperature did not decrease below a critical point (8 ° C) no loss of developmental capacity of the plants was detected. However, while new leaves were readily formed upon return to normal growth conditions (22/18 °C, day/night, in a greenhouse), net accumulation of biomass showed a lag phase of approximately one week. This delay was accompanied by a strong, irreversible inhibition of photosynthesis in the fully expanded leaves which had been exposed to the chilling treatment. When plants were subjected to temperatures below 8 ° C, survival rates decreased after three weeks at 6 ° C and irreversible damage of apical meristematic tissue occurred. Drought-hardening prior to chilling ensured survival at 6 ° C and protected the plants against meristem loss.Abreviation Chl chlorophyll Thanks are due to G.P. Telkamp for technical assistance. This research is financially supported by the Netherlands Technology Foundation (STW, Utrecht, The Netherlands), and is coordinated by the Foundation for Biological Research (BION, 's-Gravenhage, The Netherlands).     

甘草叶片渗透调节物质及蔗糖代谢相关酶对干旱胁迫的响应特性

该研究以甘草幼苗为试材,采用盆栽自然干旱方法,设计对照(CK)、轻度(LS)、中度(MS)、重度(SS)干旱胁迫处理,测定分析甘草叶片的渗透调节物质及蔗糖代谢相关酶[蔗糖磷酸合成酶(SPS)、蔗糖合成酶合成方向(Ss+)、蔗糖合成酶分解方向(Ss-)、中性转化酶(NI)、酸性转化酶(AI)、淀粉磷酸化酶(SP)]活性的变化,以探讨甘草的渗透调节特性以及糖分调节的酶学机制,揭示甘草对干旱胁迫的响应机理。结果显示:(1)随着干旱胁迫程度的加剧,甘草叶片可溶性糖、可溶性蛋白和脯氨酸含量呈逐渐增加的趋势,束缚水/自由水的比值呈先增加后降低的趋势。(2)随着干旱胁迫程度的加剧,甘草叶片蔗糖、葡萄糖、果糖含量均呈先升高后降低的趋势,但不同胁迫强度出现峰值的时间不同;其中在CK和LS干旱胁迫时蔗糖含量淀粉含量葡萄糖含量果糖含量,在MS和SS干旱胁迫时淀粉含量葡萄糖含量蔗糖含量果糖含量,表明随着干旱程度的增加,甘草叶片中蔗糖转化成了淀粉。(3)随着干旱胁迫程度的加重,甘草叶片的SPS活性呈先升高后降低的趋势,Ss活性和Inv(蔗糖转化酶)活性呈逐渐升高的趋势,SP活性呈逐渐降低的趋势;各胁迫处理的Ss+活性与CK差异不显著,而Ss-活性与CK差异显著,并且Ss-活性在各胁迫处理下远大于Ss+活性,表明甘草叶片Ss-活性在蔗糖代谢过程中占主导作用。(4)相关分析结果显示,在LS中,NI与蔗糖呈负相关关系,Ss-与淀粉呈显著正相关关系、与蔗糖呈负相关关系;在MS中,蔗糖和葡萄糖与SPS、Ss+、Ss-、NI和AI均呈正相关关系,与SP呈负相关关系;在SS中,SP和NI与蔗糖呈正相关关系,而与淀粉呈负相关关系;表明在轻度干旱处理下,Ss参加了蔗糖的分解,继而合成淀粉;在中度和重度干旱条件下,SP主要催化淀粉的分解来增加蔗糖含量以此平衡蔗糖代谢。     

Changes in Mulberry Leaf Metabolism in Response to Water Stress

A series of experiments were conducted to characterize the water stress-induced changes in the activities of RuBP carboxylase (RuBPCO) and sucrose phosphate synthase (SPS), photosystem 2 activity, and contents of chlorophylls, carotenoids, starch, sucrose, amino acids, free proline, proteins and nucleic acids in mulberry (Morus alba L. cv. K-2) leaves. Water stress progressively reduced the activities of RuBPCO and SPS in the leaf extracts, the chlorophyll content, and PS2 activity in isolated chloroplasts. Plants exposed to drought showed lower content of starch and sucrose but higher total sugar content than control plants. While the soluble protein content decreased under water stress, the amino acid content increased. Proline accumulation (2.5-fold) was noticed in stressed leaves. A reduction in the contents of DNA and RNA was observed. Reduced nitrogen content was associated with the reduction in nitrate reductase activity. SDS-PAGE protein profile showed few additional proteins (78 and 92 kDa) in the water stressed plants compared to control plants.     

Effect of Kinetin on Starch and Sucrose Metabolising Enzymes in Salt Stressed Chickpea Seedlings

Higher amylase activity in cotyledons of kinetin treated salt stressed (75 mM NaCl) chickpea (Cicer arietinum L. cv. PBG-1) seedlings, as compared to salt stressed seedlings was observed during a growth period of 7 d. The activities of acid and alkaline invertases were maximum in shoots and minimum in cotyledons under all conditions. The reduced shoot invertase activities under salt stress were enhanced by kinetin with a simultaneous increase in reducing sugar content. Kinetin increased the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in both the cotyledons and shoots of stressed seedlings. Kinetin appears to increase the turnover of sucrose in the shoots of stressed seedlings.     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Chilling, oxidative stress and antioxidant responses in shoot cultures of rice

Chilling of shoot cultures from Oryza sativa L. cv. Taipei 309, to 4 °C leads to conditions of oxidative stress. Tissue H₂O₂ was observed to increase more than fourfold by 8 d of chilling, and levels of reduced glutathione, which normally rise in growing shoot cultures at 25 °C, were considerably repressed in chilled cultures. Whilst the activity of ascorbate peroxidase in chilled shoots remained similar to the activities in control cultures at 25 °C, the most notable effects of chilling to 4 °C were the very significant loss of catalase and glutathione reductase activity. Although prior exposure of shoot cultures to abscisic acid (ABA) at 25 °C increased levels of catalase activity, such increased levels were not sustained when the pre-treated cultures were placed at 4 °C. Moreover such pre-treatment with ABA did not increase the subsequent ability of shoot cultures to grow at 4 °C.Abbreviations GSH reduced glutathione - GSSG oxidised glutathione - ABA cis-abscisic acid This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.