美洲商陆PaENO基因克隆及其表达分析

烯醇化酶(enolase,ENO)作为糖酵解途径的一个酶,还能够参与植物生长发育以及应对非生物胁迫。该研究以镉超富集植物——美洲商陆(Phytolacca americana L.)为材料,根据美洲商陆转录组数据,设计特异性引物,从叶片中克隆PaENO基因,并进行序列分析、原核表达与纯化、表达模式分析以及抗镉实验,为深入研究PaENO蛋白的酶活性质以及PaENO基因在美洲商陆应对镉胁迫过程的功能机制奠定基础。结果表明：(1)成功克隆获得PaENO基因(GenBank登录号为JN656932.1),该基因开放阅读框为1 335 bp,编码444个氨基酸。(2)多序列比对分析显示,PaENO蛋白与冰叶日中花的ENO蛋白序列一致性最高,为93.47%;系统进化分析表明,PaENO与冰叶日中花、甜菜、菠菜的亲缘关系较近,处于同一进化分支。(3)RT PCR结果显示,PaENO基因在美洲商陆叶中表达量最高为根的2.5倍,茎中表达量最低,仅为根的1/5;Cd胁迫后PaENO基因表达水平显著升高,并于2 h时PaENO基因表达量迅速升高至Cd胁迫前(0 h时)的9.54倍,Cd胁迫12 h、24 h时降低到0 h时的 3.12倍和2.89倍,说明PaENO基因参与了美洲商陆应对Cd胁迫的响应。(4)通过成功构建原核表达载体pET28a PaENO并转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后,在50 kD左右出现目的条带,然后用超声破碎大肠杆菌细胞,离心后分别取上清和沉淀,SDS PAGE检测发现,PaENO在上清和沉淀中都有表达,而且在上清中的表达量较高;使用Ni亲和层析试剂盒纯化PaENO蛋白,获得了纯化的PaENO蛋白;镉抗性实验结果初步证实,表达PaENO蛋白能够显著提高大肠杆菌对镉的抗性。研究认为,PaENO蛋白可能以转录因子(PaMBP 1)的形式在美洲商陆应对镉胁迫过程中发挥调控作用。     

Accumulation and detoxification of manganese in hyperaccumulator Phytolacca americana

Pokeweed ( Phytolacca americana ) has recently received much attention because of its ability to hyperaccumulate manganese (Mn). The internal mechanism of detoxification of Mn, however, is not fully understood. In the present study, we investigated Mn accumulation, subcellular distribution, chemical speciation and detoxification through oxalate in pokeweed. The plant accumulated excess Mn in the leaves, mainly in the water-soluble fraction, and over 80% of Mn was in a water-soluble form, while accumulation of excess Mn in the cellular organelle and membrane fraction caused phytotoxicity. In addition, pokeweed has an intrinsically high oxalate content. In all experiments, there was sufficient oxalate to chelate Mn in leaf water extracts at all different levels of Mn application. Phase analysis of X-ray diffraction detected oxalate–Mn chelate complexes, and gel chromatography further confirmed the chelation of Mn by oxalate. In conclusion, pokeweed accumulates excess Mn in the soluble fraction of leaf cells, most likely in vacuoles, in which detoxification of Mn could be achieved by chelation with oxalate.     

Effect of calcium carbonate and organic matter on the growth and concentration of iron and manganese in sorghum (Sorghum bicolor)

Summary The effect of CaCO₃ and organic matter on the availability of iron and manganese was studied on sorghum (Sorghum bicolor). Addition of calcium carbonate caused significant decrease in dry matter and grain yield. But yields tended to increase with the addition of organic matter at the rate of 15 t/ha. The iron concentration decreased with the addition of CaCO₃ at all the stages of crop growth. Like iron, concentration of manganese also decreased with the increase in added CaCO₃. Organic matter increased the iron concentration both at seedling and flowering stage. The concentration of manganese was, however, dependent on the stage of the crop.     

Expression of AtECA3 in tobacco modifies its responses to manganese, zinc and calcium

Mn- and Zn-deficiency or excess reduce plant growth and development. Engineering plants with enhanced metal tolerance and accumulation is a major goal in phytoremediation/phytostabilization. Moreover, improved growth under unfavourable mineral conditions contributes to better crop production. In this study, ECA3 cDNA from Arabidopsis thaliana, encoding a P_2A-ATPase, was introduced into Nicotiana tabacum var. Xanthi, to examine its value for modifying responses to cations, primarily Mn, Zn and Ca. AtECA3 was ectopically expressed under the CaMV 35S promoter. Transgenic and wild-type plants were tested under hydroponic conditions for their responses to a range of metal exposures (low, moderate and high concentrations), and their tolerance and accumulation were evaluated. AtECA3 expression resulted in better growth of plants at moderate levels of Mn (2 μM) in the medium and enhanced tolerance to high Mn (100 μM). Transgenic plants were also more tolerant to Ca-deficiency conditions, although they showed no differences to wild-type with respect to overall Ca levels. Transgene expression did not produce one unique pattern of Mn and Zn accumulation but instead depended on the external concentration of the particular metal supplied. Thus the enhancement of plant productivity at moderate Mn levels and increased Mn tolerance at high (toxic) Mn supply, as well as the slight increase in Ca-deficiency tolerance seen in ECA3-transformed plants indicates that this gene could be useful in plant biotechnological strategies.     

油茶微量元素铜铁锌吸收和积累特征

为弄清油茶(Camellia oleifera)对微量元素铜、铁、锌的吸收利用特征,对5年生‘长林4号’各器官的铜、铁、锌元素含量及其年变化进行了研究。结果表明,油茶植株中铁元素的含量最高,其次为锌和铜元素;单株油茶对锌、铜元素的年积累量分别为62.97 mg和22.60 mg,约为3∶1。从果实发育期至成熟期,锌元素的单株吸收积累量为40.18 mg,约占年吸收积累量的63.81%,从抽梢期至果实成熟期,铜元素的单株吸收积累量为20.04 mg,占年吸收积累量的88.67%,从休眠期至抽梢期,油茶地上部分生长所需的铜、锌元素分别有30.25%和57.90%来源于根系贮存的营养;油茶对铁元素的吸收积累则集中在抽梢期至果实发育期,单株吸收量达0.34 g。这些为指导油茶科学施肥提供了理论依据。     

Accumulation of osmolytes and osmotic adjustment in water-stressed wheat (Triticum aestivum) and maize (Zea mays) as affected by calcium and its antagonists

Maize (Zea mays) and wheat (Triticum aestivum) were water stressed for 4 days at early vegetative growth (15-day-old) using PEG-6000 (−1.0 MPa), in the presence of 1 mM CaSO₄, 50 μM Verapamil (VP; calcium channel blocker); 50 μM Trifluoperazine (TFP; calmodulin antagonist) and then put to recovery in order to investigate the changes in osmoregulation in plants having C₃ and C₄ metabolism. Accumulation of proline (Pro) and quaternary ammonium compounds (QAC's), activities of pyrroline-5-carboxylate reductase (P5CR), proline dehydrogenase (PDH), water potential (Ψ_w), osmotic adjustment (OA), relative elongation rate (RER) and electrolyte leakage (EL) were examined during stress and recovery. Maize had significantly higher accumulation of Pro while wheat showed relatively more accumulation of QAC's. The activities of P5CR and PO were also significantly higher in maize than wheat. Maize shoots under stress showed higher Ψ_w, OA, RER and less EL than wheat shoots. Upon recovery from stress, maize regained its growth and water potential faster than wheat. Ca²⁺ elevated the accumulation of osmolytes in both the plants but OA was less sensitive to it. In the presence of Ca²⁺, wheat showed significantly more accumulation of osmolytes, higher Ψ_w, RER than maize. Ca²⁺ inhibitors partially reversed the effects of calcium indicating its involvement in governing solute accumulation. The differential sensitivity of maize and wheat towards water stress may be related to variation in endogenous calcium expression and its function.     

Development and effectiveness of a soluble manganese silicate compound in controlling manganese deficiency in plants

Summary A study was made of soluble manganese silicates prepared by adding a solution of manganese sulfate to different concentrations of sodium silicate in alkaline medium. The solubility of manganese increased with increasing amounts of sodium silicate and reached its maximum at a molar Na₂SiO₃/MnSO₄ ratio between 10 and 15. At molar Na₂SiO₃/MnSO₄ ratios of 5 and more a red-brown coloured manganese silicate is formed, which is stable in the pH range of 4 to 12. Gel chromatographic analysis indicated that this compound contains 1 molecule Mn per 15 molecules SiO₂. At a molar Na₂SiO₃/MnSO₄ ratio of 15 the yield of soluble manganese could be raised to 98.5% of the theoretical amount by preventing the rapid oxidation of the Mn²⁺-ions in the alkaline solution. A pot experiment with oats was conducted to evaluate the effectiveness of the red-brown manganese silicate in controlling manganese deficiency. The effect of the Mn-carrier on Mn-content and yield of grain and on the colour scores was compared with that of Mn-EDTA, Mn-DTPA and MnSO₄·4H₂O. The manganese silicate was found to be a more effective manganese source than Mn-EDTA, Mn-DTPA or MnSO₄·4H₂O.     

The interaction of manganese ions with DNA

We present the structure of the duplex formed by a fragment of auto-complementary DNA with the sequence d(CGTTAATTAACG). The structure was determined by X-ray crystallography. Up to date it is the first structure presenting the interaction between a DNA oligonucleotide and manganese ions. The presence of Mn²⁺ creates bonds among the N7 atom of guanines and phosphates. These bonds stabilize and determine the crystallographic network in a P3₂ space group, unusual in oligonucleotide crystals. The crystal structure observed is compared with those found in the presence of Mg²⁺, Ca²⁺ and Ni²⁺, which show different kinds of interactions. The double helices show end-to-end interactions, in a manner that the terminal guanines interact with the minor groove of the neighboring duplex, while the terminal cytosines are disordered. We have chosen this sequence since it contains a TTAA repeat. Such repeats are very rare in all genomes. We suggest that this sequence may be very susceptible to the formation of closely spaced thymine dimers.     

灵山湾国家森林公园垂序商陆土壤种子库时空动态及其对防除措施的响应

土壤种子库是植物潜在的种群,对生态系统稳定和未来植被的结构、功能有着举足轻重的影响。垂序商陆(Phytolacca americana)的入侵已对山东省胶南市灵山湾国家森林公园的森林生态系统构成严重危害。为调查该物种在公园内的潜在扩散能力,于2009—2014年调查了刺槐(Robinia pseudoacacia)林、黑松(Pinus thunbergii)林、刺槐黑松混交林和紫穗槐(Amorpha fruticosa)林(灌丛)4种林分类型,以及2009年采用物理和化学控制入侵危害严重的刺槐林内的土壤种子库。结果表明:垂序商陆种子在4种林分中均有发现,在刺槐、黑松林内表现为由林缘至林内逐步降低的趋势。垂序商陆种子只存在于枯落物层和5 cm以上的土壤中,随土层深度的增加而减少,枯落物层土壤种子库由林缘到林内呈递减趋势。与2009年相比,2014年黑松林和灌丛中平均种子库密度分别降低46%和44%,刺槐林和混交林分别增加299%和355%。垂序商陆种子发芽率在10%以下,随土层深度的增加而依次降低。采用物理、化学控制措施的刺槐林内垂序商陆的更新量、结实量、种子库含量均明显高于紫穗槐林的。调查结果表明垂序商陆土壤种子库具有明显的空间异质性,并且保持了较强的繁殖潜力,营造紫穗槐灌丛可以有效控制垂序商陆的扩散,可为经营沿海防护林提供理论指导。     

Crystal structure of the mononuclear complex [Mn(mesalim)2Cl] and electrochemical studies of manganese complexes of the ligand Hmesalim

The complex [Mn(mesalim)₂Cl] (1), (Hmesalim = methyl salicylimidate) has been synthesized and fully characterized. The manganese(III) complex is formed by the reaction of the ligand Hmesalim with manganese(II) chloride. Complex 1 is mononuclear and crystallizes in the space group . Electrochemical studies were performed for complex 1, as well as for the related complexes [Mn(mesalim)₂(OAc)(MeOH] · MeOH (2) and [Mn₂(etsalim)₄(Hetsalim)₂](ClO₄)₂ (3), (Hetsalim = ethyl salicylimidate). The complexes display intricate oxidation-reduction behaviour, and coulometric analyses in combination with electrochemical analyses have been used to understand the electron transfer mechanisms occurring at the electrodes.     

Crystal structures and magnetic properties of two complexes synthesized from manganese and halogenophenyl-substituted nitronyl nitroxide

One-dimensional complex (1), [Mn(hfac)₂(NITPhF)]₂ and one binuclear radical complex (2), [Mn(hfac)₂(NITPhBr)]_∞ have been synthesized. Here hfac stands for hexafluoroacetylacetonate, NITPhF for 2-(4′-fluorophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and NITPhBr for 2-(4′-bromophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide). All the two precursors were prepared and characterized by single-crystal X-ray diffraction analysis, IR, and magnetic analysis. In complex 1, the NITPhF radical acts as a bridge ligand linking two Mn(II) ions through the oxygen atom of the N-O group to form cyclic dimer. The dimers further connect two oxygen atoms of uncoordinated nitroxides of two adjacent radicals and yield one-dimensional chain sections. Instead in complex 2, the Mn(II) ions are bridged by the NITPhBr radicals through their N-O groups giving infinite one-dimensional chains. Magnetic susceptibility measurements indicate that both complex 1 and 2 behave ferrimagnetically. The Mn(II) ions interact antiferromagnetically with the direct bonding nitroxide group of the radicals although the structures of two complexes are different. The magnetic behaviors can be satisfactorily explained on the basis of the structural data.     

A seven-coordinate manganese(II) complex formed with the single heptadentate ligand N,N,N′-tris(2-pyridylmethyl)-N′-(2-salicylideneethyl)ethane-1,2-diamine

The new disymmetric ligand N,N,N′-tris(2-pyridylmethyl)-N′-(2-salicylideneethyl)ethane-1,2-diamine (LH) has been synthesized in the search of a novel type of manganese complex to mimic the active site of the water-oxidizing enzyme in photosystem II. The complex [Mn(II)L]ClO₄ has been obtained and characterized by X-ray diffraction techniques. It crystallizes in the monoclinic space group Pn with the following unit cell parameters: a=10.164(3), b=10.122(4), c=14.166(5) Å, β=93.48(2)° and Z=2. The manganese ion is heptacoordinated with the coordination being achieved by only one ligand; it is bonded to the oxygen atom of the phenolate group in an axial position, the imino and the three pyridine nitrogen atoms in an equatorial position and the two amine atoms in a pseudo-axial position. The coordination polyhedron is best described as a distorted monocapped trigonal prism. This structure was compared with the seven-coordinated Mn(II) complexes deposited in the Cambridge Structured Database (CSD). The redox potential of the Mn(III)/Mn(II) couple was determined by cyclic voltammetry.     

Decreasing expression of alpha1C calcium L-type channel subunit mRNA in rat ventricular myocytes upon manganese exposure

Manganese is an essential trace element found in many enzymes. As it is the case of many essential trace elements, excessive level of manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to understand the mechanism of manganese toxicity in the heart, the effects of manganese on isolated rat ventricular myocytes were studied. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50 microM Mn2+ and 100 microM Mn2+ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover, Mn2+ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. Investigation by RT-PCR showed that the mRNA expression of alpha1C/Cav1.2 treated with manganese was decreased depending on its concentration, while the mRNA expression of alpha1D/Cav1.3 was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM), and the results showed that Mn2+ could elicit a slow and continuous increase of [Ca2+]i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, downregulate the mRNA expression of alpha1C/Cav1.2, and thus causing long-lasting molecular changes of L-type calcium channel which have probably been triggered by overloading of calcium in myocytes.     

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae

In a search for components involved in Mn²⁺ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn²⁺ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn²⁺ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co²⁺, Ni²⁺, Zn²⁺, and Cu²⁺. The Mn²⁺ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn²⁺ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn²⁺ efflux. We propose that Hip1p is involved in Mn²⁺ transport, carrying out a function related to Mn²⁺ export. Received: 9 January 1998 / Accepted: 4 May 1998     

Hg2+在中华绒螯蟹幼蟹鳃内的积累及其对组织结构的影响

采用生态学单因子梯度实验,研究不同浓度汞(Hg2 )的水环境中中华绒螯蟹(Eriocheir sinensis)幼蟹鳃内汞积累情况及对鳃组织结构的影响。结果表明,中华绒螯蟹幼蟹的鳃能够从水环境中迅速积累汞,Hg2 浓度为10、50、100μg/L组的水环境中幼蟹鳃内汞含量分别为4.56、25.53、46.51μg/g干重,均显著高于对照组幼蟹鳃内汞含量1.68μg/g干重(P<0.05);水环境Hg2 浓度为200、300μg/L组的幼蟹鳃内汞含量达到了183.75和283.36μg/g干重,对水体Hg2 蓄积倍数达到了910.35和939.60。光镜观察低浓度组汞能够诱导鳃组织结构发生变化,如上皮细胞肿胀、鳃叶厚度增加、端部膨胀等;高浓度组则会导致鳃叶上皮细胞崩解,鳃组织结构完全破坏。以统计学方法分析了鳃内汞含量同水体汞浓度的相关性,结果表明,两者之间存在明显的相关性,并能够在组织形态上有所体现。鳃是中华绒螯蟹最容易接触水体Hg2 的器官,同时也是蓄积汞的主要器官之一,水体高浓度的Hg2 能够对鳃的组织结构造成明显的破坏,严重影响幼蟹存活率。     

The synthesis and reactions of A-ring aromatic steroid complexes of manganese tricarbonyl

Treatment of the A-ring aromatic steroids estrone 3-methyl ether and β-estradiol 3, 17-dimethyl ether with Mn(CO)₅⁺BF₄⁻ in CH₂Cl₂ yields the corresponding [(steroid)Mn(CO)₃]BF₄ salts 1 and 2 as mixtures of and β isomers. The X-ray structure of [(estrone 3-methyl ether)Mn(CO)₃]BF₄ · CH₂Cl₂ (1) having the Mn(CO)₃ moiety on the side of the steroid is reported: space group P2₁ with a=10.3958(9), b=10.9020(6), c=12.6848(9) Å, β=111.857(6)°, Z=2, V=1334.3(2) ų, _calc=.481 cm⁻³, R=0.0508, and wR=0.0635. The molecule has the traditional ‘piano stool’ structure with a planar arene ring and linear Mn---C---O linkages. The nucleophiles NaBH₄ and LiCH₂C(O)CMe₃ add to [(β-estradiol 3,17-dimethyl ether)Mn(CO)₃]BF₄ (2) in high yield to give the corresponding - and β-cyclohexadienyl manganese tricarbonyl complexes (3). The nucleophiles add meta to the arene -OMe substituent and exo to the metal. The and β isomers of 3 were separated by fractional crystallization and the X-ray structure of the β isomer with an exo-CH₂C(O)CMe₃ substituent is reported (complex 4): space group P2₁2₁2₁ with a=7.5154(8), b=15.160(2), c=25.230(3) Å, Z=4, V=2874.4(5) ų, _calc=1.244 g cm⁻³, R=0.0529 and wR2=0.1176. The molecule 4 has a planar set of dienyl carbon atoms with the saturated C(1) carbon being 0.592 Å out of the plane away from the metal. The results suggest that the manganese-mediated functionalization of aromatic steroids is a viable synthetic procedure with a range of nucleophiles of varying strengths.     

Seasonal variation of calcium,magnesium, potassium,and manganese contents in xylem sap of beech(Fagus sylvatica L.) in a 35-year-old limestone beech forest stand

Summary In a 35-year-old calcareous beech forest stand five beech trees (Fagus sylvatica L.) were felled every 2 weeks, and xylem sap was obtained by means of water displacement from the lowest trunk sections, each 100 cm in length. From mid-October 1988 to mid-October 1989 a total of 130 trees were investigated. The seasonal variations of the Ca, Mg, K and Mn contents, as well as those of pH, show four characteristic phases. Additionally, distribution of the mineral contents along the trunk was studied in four trees. The seasonal increase and decrease of xylem sap mineral contents along the trunk is shown for the characteristic phenophases. The Ca, Mg, K, and Mn contents of xylem saps were determined by means of atomicabsorptionspectrophotometry.     

贵州苏铁种子钙形态特性分析

通过测定贵州苏铁新鲜成熟种子中不同组织中总钙和不同形态类型钙物质含量,探索贵州苏铁种子发育过程中对钙的储存形式和分布规律。结果表明贵州苏铁新鲜成熟种子中总钙含量为外种皮>中种皮>胚;钙主要集中在外种皮并且主要以HCl钙形态存在,其含量高达470.8 mg/kg,占外种皮钙形态的56.3%,以及组织和整个种子的24.6%和11.4%,HCl钙形态在种子胚的发育过程中主要起到源和库的作用;中果皮和胚主要积累的是水溶性的有机钙,含量分别为70.2 mg/kg和70.6 mg/kg,对参与种子的结构和胚的发育起到积极作用。另外,贵州苏铁种子中可溶性蛋白质和可溶性糖含量与外种皮高钙含量和高钙形态含量密切相关,而大分子物质的积累对促进贵州苏铁适应高钙质环境具有积极意义。     

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Summary Crystal idioblasts are cells which are specialized for accumulation of Ca²⁺ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of⁴⁵Ca²⁺ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca²⁺ sinks, accumulating large amounts of Ca²⁺ within the vacuole as crystals. The pattern of addition of Ca²⁺ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,⁴⁵Ca²⁺ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca²⁺ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.Abbreviation SEM scanning electron microscopy     

Influence of calcium oxide level and time of exposure to sugarcane on in vitro and in situ digestive kinetics

These experiments were carried out to evaluate, using in vitro and in situ techniques, the effects of three inclusion levels of calcium oxide (0, 5, and 10 g/kg of sugarcane fresh matter) and four exposure times (0, 24, 48, and 72 h) of sugarcane to calcium oxide on the chemical composition and digestive kinetic parameters of sugarcane. The treatments were arranged in a 3 by 4 factorial design. Freshly-cut sugarcane (whole plant) was treated with calcium oxide and separated into 12 piles inside a barn to prevent direct exposure to sunlight, rain, and wind. Every day, before and after animal feeding, the calcium oxide was proportionally hand-mixed with approximately 150 kg of freshly-cut sugarcane to make up the dietary treatments. The lowest (Ti) and greatest (Ts) temperature and pH of the treated sugarcane piles were measured immediately before and after sampling, respectively. The ether extract (EE) and DM were not affected (P>0.05) by either exposure time or inclusion level. However, CP increased linearly (P=0.01) and OM decreased linearly (P<0.0001) as the exposure time and calcium oxide inclusion level increased. Interactions between inclusion level and exposure time on DM, OM, CP, EE, Ti, and Ts were not observed. However, significant interactions were detected for non-fibre carbohydrate (NFC), neutral detergent fibre (aNDF), and pH. A quadratic effect of exposure time on the Ti and Ts was observed (P=0.001 and P=0.001, respectively). The maximum temperature was reached with approximately 51 h of exposure time. Calcium oxide positively affected the insoluble potentially digestible fraction of sugarcane DM and aNDF (P=0.001 and P=0.001, respectively), and the indigestible fraction of sugarcane aNDF (P=0.001). Interactions between inclusion level and exposure time on soluble and indigestible fractions of sugarcane DM (P=0.0001 and P=0.01, respectively) were found. However, no interactions (P>0.27) were found between inclusion level and exposure time on aNDF digestive kinetic parameters. The fractional digestion rate (kd) of sugarcane DM and aNDF was not influenced by treatments (P>0.05). The mean values of kd for sugarcane DM and aNDF were 0.0235 and 0.0215/h, respectively. The gas production kinetics parameters were not affected (P>0.05) by treatments. In conclusion, the inclusion of calcium oxide improved the in situ potentially digestible fraction of sugarcane DM and aNDF; however, it did not influence the fractional digestion rate. No effects were observed on the in vitro digestive kinetic parameters.