Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2 fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9–mCherry fusion protein was detected 6 h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9–mCherry secretion, demonstrating the utility of this method in a biological experiment.     

Adaptability of tomato and pepper leaves to changes in photoperiod: Effects on the composition and function of the thylakoid membrane

The adaptability of the thylakoid membrane to extended photoperiod (from natural to 24 h) was studied using a photoperiod-sensitive species ( Lycopersicon esculentum Mill. cv. Trend) and a non-photoperiod-sensitive species ( Capsicum annuum L. cv. Delphin). Our results have shown that thylakoid membranes of both species adapt to an extended photoperiod by increasing their photosystem II to photosystem I ratio (PSII/PSI) in order to provide a more balanced energy distribution between both photosystems to improve quantum yield. In tomato plants, these results correspond with a lower chlorophyll (Chl) a/b ratio, a decrease in Chl associated with PSI light-harvesting chlorophyll a/b protein complexes and with an increase in Chl associated with PSII light-harvesting chlorophyll a/b protein complexes. In spite of these changes, the electron transport capacity through PSII and PSI per unit of Chl and the light saturation point of PSII remained unchanged. The inability of tomato plants to use supplemental light for an extended photoperiod is not the result of photoinhibitory conditions. In pepper plants a significant increase in electron transport capacity and in the light saturation point of PSII was found. There was a significant increase in CO₂ assimilation when the light period was increased from 12 to 24 h. In contrast to tomato, pepper plants adapt to a 24-h photoperiod by increasing their carboxylation capacity which is accompanied by an increase in electron transport capacity and the light saturation point.     

Nature of the Carbohydrate Side Chains and Their Linkage to the Protein in Chicken Egg White Ovomucin

Some proteolytic digests of chicken egg white ovomucin were fractionated and characterized. It was shown that there are at least three types of carbohydrate side chains in ovomucin; a chain composed of galactose, galactosamine, sialic acid and sulfate in a molar ratio of about 1: 1: 1: 1, a chain composed of galactose and glucosamine in a molar ratio of about 1:1, and a chain composed of mannose and glucosamine in a molar ratio of about 1:1. It was also shown that the carbohydrate side chain composed of galactose, galactosamine, sialic acid and sulfate is linked O-glycosidically to serine or threonine in the protein core of ovomucin.     

The function of carotenoids during chloroplast development. V. Correlation between carotenoid content, ultrastructure and chlorophyll b to chlorophyll a ratio

The ultrastructural effect of carotenoid deficiency in wheat ( Triticum aestivum L. ) was studied after adding the herbicide SAN-9789 to the growth medium. The presence of SAN-9789 (28 mg I^-1) resulted in an almost complete absence of carotenoids. For plants grown in darkness the lack of carotenoids was accompanied by a reduction in partitions between primary thylakoids as well as a change in appearance of the plastoglobuli from small and black (osmiophilic) to large and greyish white (less osmiophilic). When plants were grown in weak red light (16 mW m^-2), the presence of SAN-9789 also resulted in an almost complete absence of grana, a decrease in the ratio of chlorophyll b /chlorophyll a from 0.25 to 0.1, and an almost complete absence of prolamellar bodies. The greatest differences in carotenoid content, in amount of grana, in chlorophyll b /chlorophyll a ratio, and in number of prolamellar bodies, all occurred between 0.28 and 2.8 mg I^-1 of SAN-9789. The change in appearance of plastoglobuli occurred at a lower SAN-9789 concentration, between 0.028 and 0.28 mg I^-1. The ultrastructural responses to the SAN-9789 treatment could be related to the effect on the carotenoid synthesis in different ways. The disappearance of thylakoid partitions was connected to a structural role of carotenoids, while the disappearance of the prolamellar bodies was dependent on the photoprotective role of carotenoids. The change in shape and size of plastoglobuli was not correlated to the presence of carotenoids. However, a connection to the accumulation of the carotenoid precursors is still possible.     

Changes in the chlorophyll and carotenoid contents in the leaves of steppe plants along a latitudinal gradient in South Ural

We studied the content of chlorophylls and carotenoids in the leaves of steppe plants of South Ural growing along a latitudinal gradient from southern steppe to forest-steppe. The content of chlorophylls (a + b) was 5–6 mg per 1 g of the leaf dry weight and did not depend on the latitude, whereas the content of carotenoids in the leaves increased northward from 1.0 to 1.5 mg/g dry wt. At the same time, the greatest changes occurred in the ratios between the forms of pigments: the chlorophyll a/b ratio increased from 1.8 to 2.8, and the chlorophyll/carotenoid ratio decreased from 5.6 to 3.5. The obtained results indicate that adaptation of the pigment apparatus of steppe plants growing along the latitudinal gradient occurs due to the transformation of the light-harvesting complex.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

自控玻璃温室环境气象条件对甜椒叶龄和果实生长的影响

根据在自控玻璃温室内甜椒试验资料和同期的环境气象资料 ,运用数理统计分析方法 ,建立了甜椒叶龄模式、果实生长和果重模式。叶龄生长符合植物生长模型 ,影响叶龄生长的主要环境气象因子在打顶前是日平均气温 13～ 2 2℃的有效积温 ,打顶后是日平均气温 13～2 5℃的有效积温。影响甜椒果实膨大的主要环境气象因子是座果后的日平均气温 16～ 2 3℃的有效积温、日照、温度日较差和CO2 浓度 ,甜椒果实膨大与环境气象因子间的关系呈对数形式。果实生长期的长短与日平均气温 14～ 2 3℃的有效积温、日照、温度日较差和CO2 浓度有关。果实的重量与日平均气温 14～ 2 3℃的有效积温、温度日较差和CO2 浓度有关。开花前 2d至开花后 3d的日平均光照小于 1 5× 10 41x ,开花后 3～ 12d日较差小于 6 5℃ ,开花前 18d至开花后 3d日平均相对湿度大于 82 %时阴果明显增多     

Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin

Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm β : ε lycopene cyclase activity favours the β‐carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene β‐cyclase) and therefore accumulating extraordinary levels of β‐carotene. Here, we demonstrate that introgressing the transgenic mini‐pathway into wild‐type yellow endosperm varieties gives rise to hybrids in which the β : ε ratio is altered additively. Where the β : ε ratio in the genetic background is high, introgression of the mini‐pathway allows zeaxanthin production at an unprecedented 56 μg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.     

气候与土壤对烤后烟叶类胡萝卜素和表面提取物含量的影响

选择贵州生态条件差异较大的威宁县、开阳县和天柱县进行客土田间小区试验,以云烟85为材料,探讨气候与土壤对烤后烟叶类胡萝卜素和表面提取物含量的影响.结果表明:类胡萝卜素和表面提取物含量受生态条件的影响较大,地域性差异显著,打破犁底层耕作方式无明显作用.类胡萝卜素含量有随海拔高度增加而升高的趋势,但海拔高度的影响存在阈值效应;清香型烟叶的表面提取物含量明显高于中间香型.不同生态因子的影响程度明显不同,气候是关键生态影响因子.类胡萝卜素含量气候间的变异度是土壤的2-3倍,气候对β-胡萝卜素与叶黄素含量变异的贡献率分别为74％和64％,土壤贡献率分别为9％和12％.影响较大的前3个气候因子为大田生长前期的累积日照时数和累积降水量、成熟期的相对湿度,前3个土壤因子为有效钾、pH值和有效磷.表面提取物含量气候间的变异度是土壤的3-4倍,气候对腺毛分泌物与烷烃类蜡质含量变异的贡献率分别为61％和63％,土壤贡献率分别为13％和7％.影响腺毛分泌物含量的前3个气候因子为大田生长前期的平均气温、成熟期的相对湿度和≥10℃积温,前3个土壤因子为有效钾、有效铜含量和pH值.烷烃类蜡质含量的前3个气候影响因子为大田生长前期的累积降水量和平均气温、成熟期的平均气温,前3个土壤因子为有效锰、有效磷含量和阳离子交换量.土壤因子对烤后烟叶类胡萝卜素含量部位特征的影响较气候因子突出,气候因子对表面提取物含量部位特征的影响较土壤因子明显.     

Homologs of the Genes for Receptor-Like Kinase Proteins Conferring Plant Resistance to Pathogens: Resistance Gene Homologs in Solanum Species

Direct amplification of the genomic DNA from cultivated and wild Solanum species was used to synthesize three groups of NBS-LRR homologs of the genes which encode the pathogen-recognizing receptor-like serine/threonine kinases (RLK): (1) the NBS-kinase regions homologous to the arabidopsis RPS2 gene, the tobacco N gene, and the flax L6 gene (the corresponding GenBank accession nos. U14158, U15605, and U27081); (2) full-size sequences homologous to the Pto gene of Lycopersicon pimpinellifolium (AF220602); and (3) LRR regions homologous to potato genesGpa2/Rx1 (AJ249449 and AJ011801) and the tomato gene Mi1 (AF091048). The nucleotide and deduced amino acid sequences of the cloned fragments of the genes and pseudogenes were compared to the already known genes and their homologs within the family Solanaceae.     

Cytokinin-induced changes in the chlorophyll content and fluorescence of in vitro apple leaves

Cytokinins (CKs) are one of the main regulators of in vitro growth and development and might affect the developmental state and function of the photosynthetic apparatus of in vitro shoots. Effects of different cytokinin regimes including different types of aromatic cytokinins, such as benzyl-adenine, benzyl-adenine riboside and 3-hydroxy-benzyladenine alone or in combination were studied on the capacity of the photosynthetic apparatus and the pigment content of in vitro apple leaves after 3 weeks of culture. We found that the type of cytokinins affected both chlorophyll a and b contents and its ratio. Chlorophyll content of in vitro apple leaves was the highest when benzyl-adenine was applied as a single source of cytokinin in the medium (1846–2176 μg/1 g fresh weight (FW) of the leaf). Increasing the concentration of benzyl-adenine riboside significantly decreased the chlorophyll content of the leaves (from 1923 to 1183 μg/1 g FW). The highest chl a/chl b ratio was detected after application of meta-topolin (TOP) at concentrations of 2.0 and 6.0 μM (2.706 and 2.804). Chlorophyll fluorescence was measured both in dark-adapted (F_v/F_m test) and in light-adapted leaf samples (Yield test; Y(II)). The maximum quantum yield and efficiency of leaves depended on the cytokinin source of the medium varied between 0.683 and 0.861 (F_v/F_m) indicating a well-developed and functional photosynthetic apparatus. Our results indicate that the type and concentration of aromatic cytokinins applied in the medium affect the chlorophyll content of the leaves in in vitro apple shoots. Performance of the photosynthetic apparatus measured by chlorophyll fluorescence in the leaves was also modified by the cytokinin supply. This is the first ever study on the relationship between the cytokinin supply and the functionability of photosystem II in plant tissue culture and our findings might help to increase plantlet survival after transfer to ex vitro conditions.     

The content of photosynthetic pigments and the light conditions in the fruits and leaves of sweet pepper

Investigations were carried out on the fruits of sweet pepper at its two development stages: on green fruits, on mature red and yellow fruits and on leaves. The content of the photosynthetic pigments and the optical properties were examined. In the green fruits when compared with leaves the content of the photosynthetic pigments is smaller by 40 to 50 % and the value of the ratio: chlorophyll a/b is lower. Chlorophyll is absent in mature fruits, while the content of carotenoids is a few times higher. The optical properties of green fruits and of the leaves in the PAR (photosynthetically active radiation) range are the same. In the range 700–1100 nm the green fruits show smaller reflectance and transmittance and a few times greater absorptance of irradiation which contributes to the warming up of the seed bag, while small absorptance of leaves in this range protects them against overheating. In mature fruits, in the PAR range, the reflectance and transmittance are higher, while the absorptance of irradiation in comparison with that of green fruits is smaller. In the range 700 – 1100 nm the changes are rather small and refer to the increase of reflectance and reduction of absorptance, while the transmittance of irradiation remains unchanged.     

光质对烟叶光合特性、类胡萝卜素和表面提取物含量的影响

以烤烟云烟87为材料,在烟株团棵期和打顶期用透光率相近的有色薄膜分别对其进行遮光处理直到采收结束,研究光质对烟叶光合特性、类胡萝卜素和表面提取物含量的影响.结果表明:烟叶生育前期各光质的整体光合性能依次为红光＞自然光＞白光＞蓝光＞黄光,后期蓝光的作用逐渐凸显.烤后烟叶β-胡萝卜素和叶黄素含量之间呈极显著正相关,且β-胡萝卜素含量明显高于叶黄素含量.团棵期增加红光,打顶期补充蓝光,有利于提高烤后烟叶β-胡萝卜素和叶黄素含量.光质对腺毛分泌物中的β-西柏三烯二醇影响最大,对降茄二酮影响最小.烷烃类蜡质成分中的三十一烷、三十三烷和异三十三烷含量受光质影响较大,异三十二烷和二十九烷含量受光质影响较小.相对于腺毛分泌物和烷烃类蜡质而言,光质对新植二烯影响较小.黄膜处理烤后烟叶的腺毛分泌物及其降解产物总量最高,光质对新植二烯和烷烃类蜡质含量的影响存在多种变化.红光和蓝光对烟叶表面提取物的影响效应主要表现在烟株生长前期,黄光和白光的影响效应主要体现在烟株生长后期.烟株生长前期增加红光比例,有利于增加烤后烟叶新植二烯和烷烃类蜡质成分积累;烟株生长后期补充黄光光质有助于提高烤后烟叶腺毛分泌物含量和表面提取物总量.     

Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein

We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed. Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts. Moreover, FP4 appears to be unprocessed. This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide. Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle. Furthermore, both FP24 and FP53 appear to be processed. However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.     

Changes in the chlorophyll content of grape leaves could provide a physiological index for responses and adaptation to UV‐C radiation

Stilbenes are a group of phytoalexins that play an important role in grapevine (Vitis) basal immunity and can be induced by biotic and abiotic stresses. The levels of chlorophylls, the main pigments in plant cells, can also indicate the tolerance of plants to various stresses. Here, the response of different grapevine genotypes to UV‐C radiation treatment was tested and the abundance of chlorophyll in the Hoe29 and Ke53 genotypes was observed to increase significantly within 6 h of UV‐C treatment. Conversely, chlorophyll levels decreased markedly in the Augster Weiss and Müller–Thurgau genotypes. Furthermore, stilbene abundance increased substantially in the Hoe29, Ke53, Ke83 and Pinot Blanc genotypes, but increased only slightly in Augster Weiss and Müller–Thurgau. The expression of resveratrol synthase, which encodes a key enzyme in the stilbene synthesis pathway, increased in Hoe29, Ke53, Ke83 and Pinot Blanc following UV‐C treatment, in a manner consistent with stilbene accumulation. In addition, we observed that reactive oxygen species (ROS) provide a key trigger in physiological responses and changes in secondary metabolite contents. In summary, the results from this study support a link between ROS, chlorophyll levels and genetic diversity for stilbene abundance in different grape genotypes, providing insights into mechanisms for plant physiological and biochemical responses and adaptations to stress.     

Analysis of quantitative trait loci affecting chlorophyll content of rice leaves in a double haploid population and two backcross populations

Chlorophyll content, one of the most important physiological parameters related to plant photosynthesis, is usually used to predict yield potential. To map the quantitative trait loci (QTLs) underlying the chlorophyll content of rice leaves, a double haploid (DH) population was developed from an indica/japonica (Zhenshan 97/Wuyujing 2) crossing and two backcross populations were established subsequently by backcrossing DH lines with each of their parents. The contents of chlorophyll a and chlorophyll b were determined by using a spectrophotometer to directly measure the leaf chlorophyll extracts. To determine the leaf chlorophyll retention along with maturation, all measurements were performed on the day of heading and were repeated 30 days later. A total of 60 QTLs were resolved for all the traits using these three populations. These QTLs were distributed on 10 rice chromosomes, except chromosomes 5 and 10; the closer the traits, the more clustering of the QTLs residing on common rice chromosomal regions. In general, the majority of QTLs that specify chlorophyll a content also play a role in determining chlorophyll b content. Strangely, chlorophyll content in this study was found mostly to be lacking or to have a negative correlation with yield. In both backcross F₁ populations, overdominant (or underdominant) loci were more important than complete or partially dominant loci for main-effect QTLs and epistatic QTLs, thereby supporting previous findings that overdominant effects are the primary genetic basis for depression in inbreeding and heterosis in rice.     

CO2和O3浓度倍增及其复合作用对大豆叶绿素含量的影响

利用开顶箱(OTC)法研究了在CO2和O3浓度倍增及其复合作用下,大豆叶片叶绿素含量及叶绿素a／b值的变化规律。结果表明,不同生育时期大豆叶片中叶绿素含量不同,Chla、Chlb和ChlT都表现出低．高一低的趋势,而且不同处理间变化不同步。不同处理间比较,O3处理的植株叶绿素含量下降最为明显,其次是复合处理的影响,而CO2浓度倍增对提高叶片叶绿素含量有一定的作用。Chla/b呈下降趋势,受CO2倍增影响最明显,有利于提高作物的光合性能。     

Regulation of photosystem stoichiometry,chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure

The structural-functional organization of higher plant chloroplasts has been investigated in relation to the particular light conditions during plant growth. (1) Light intensity variations during growth caused changes in the $\text{Chl}\text{a}\text{Chl}\text{b}$ ratio, in the light-saturated uncoupled rates of electron transport to a Hill oxidant and in the distribution of the chloroplast volume between the membrane and stroma phases. (2) Light quality differences during growth had an effect on the PS II/PS I reaction center ratio and on the chloroplast membrane phase differentiation into grana and stroma thylakoids. Plants grown under far-red-enriched (680–710 nm) illumination contained higher (20–25%) amounts of PS II and simultaneously lower (20–25%) amounts of PS I reaction centers. They also showed a higher grana density along with thicker grana stacks in their chloroplasts. (3) The size of the light-harvesting antenna pool of PS II centers was estimated from the fluorescence time course of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts and was found to be fairly constant (±10%) in spite of the variable PS II/PS I reaction center ratio. The results are compatible with the hypothesis that the structural entities of grana facilitated the centralization and relative concentration increase of a certain group of PS II reaction centers.     

Effect of light and benzyladenine on dark-treated growing rice leaves (Oryza sativa). I. Changes in chlorophyll content and catalase activity

Light- and benzyladenine-induced reversal of the changes in chlorophyll content and catalase activity were studied in the attached first leaf of Oryza sativa L. cv. Bala, kept in darkness for different periods before maturation. Dark treatment caused a decrease in chlorophyll content and catalase activity at all times. Light treatment of dark-incubated seedlings at different periods before maturation reversed the dark-induced effect on chlorophyll content, catalase activity and dry weight and also caused a further rise in chlorophyll content compared to initial values. In darkness, the application of benzyladenine replaced the light effect in maintaining catalase activity. Chlorophyll content was also maintained by initially applied benzyladenine. Benzyladenine did not promote the photoinduced maintenance and increase in chlorophyll content and catalase activity at any time. Treatment with hydrogen peroxide, glycolate and amizol resulted in an accelerated chlorophyll breakdown and had varied effects on catalase levels. Chlorophyll decrease due to peroxide accumulation was to some extent reversible by benzyladenine, but the hormone had no effect on the peroxide-induced decrease in catalase activity. Development of catalase is light dependent. Benzyladenine stabilises the enzyme but has no effect on its synthesis.