Characterization of a biodegradable starch based film. Application on the preservation of fresh spinach

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia - UL

Effects of plant density and the number of emitters on plant growth and nitrate concentration in spinach cultivated in substrate

The effects of plant density and the number of emitters per Styrofoam box on plant growth and nitrate (NO3-) concentration were evaluated in spinach (Spinacia oleracea L. cv. Tapir). Spinach seedlings were transplanted at 45 days after emergence into Styrofoam boxes filled with the substrate and were grown during winter in an unheated greenhouse with no supplemental lighting. The experiment was carried out with four treatments, including two plant densities (160 and 280 plants/m2) and two number of emitters per Styrofoam box (4 and 8 emitters). Each planting box was irrigated daily and fertigated with a complete nutrient solution. Shoot dry weight was not affected by plant density. However, yield increased with plant density and emitter number. Leaf-blade NO3- concentration was not affected by the interaction between plant density and number of emitters, but petioles NO3- concentration was greater in treatment with 160 plants/m2 and 8 emitters. Although leaf-blade NO3- concentration was not affected by plant density, it decreased with the number of emitters. On the other hand, petiole NO3- concentration was not affected by plant density or number of emitters. Leaf-blade NO3- concentration ranged from 3.2 to 4.1 mg/g fresh weight, occurring the highest value in the treatment with 280 plants/m2 and 4 emitters. Petiole NO3- concentration ranged from 3.5 to 5.3 mg/g fresh weight, values that were higher than allowed by EU regulation.

Effects of substrate type on plant growth and nitrogen and nitrate concentration in spinach

Abstract The effects of three commercial substrates (a mixture of forest residues, composted grape husks, and white peat, black peat and coir) on plant growth and nitrogen (N) and nitrate (NO3) concentration and content were evaluated in spinach (Spinacia oleracea L. cv. Tapir). Spinach seedlings were transplanted at 45 days after emergence into Styrofoam boxes filled with the substrates and were grown during winter and early spring in an unheated greenhouse with no supplemental lighting. Each planting box was irrigated daily by drip and fertilized with a complete nutrient solution. The NO3 content of the drainage water was lower in coir than in the other substrates. However, shoot NO3 concentration was not affected by substrate type, while yield and total shoot N and NO3 content were greater when plants were grown in peat than in the mixed substrate or the coir. Leaf chlorophyll meter readings provided a good indication of the amount of N in the plants and increased linearly with total shoot N. Keywords Spinacia oleracea; chlorophyll meter; coir; peat; soilless culture systems

Effects of substrate type on plant growth and nitrogen and nitrate concentration in spinach.

The effects of three commercial substrates (a mixture of forest residues, composted grape husks, and white peat, black peat and coir) on plant growth and nitrogen (N) and nitrate (NO3) concentration and content were evaluated in spinach (Spinacia oleracea L. cv. Tapir). Spinach seedlings were transplanted at 45 days after emergence into Styrofoam boxes filled with the substrates and were grown during winter and early spring in an unheated greenhouse with no supplemental lighting. Each planting box was irrigated daily by drip and fertilized with a complete nutrient solution. The NO3 content of the drainage water was lower in coir than in the other substrates. However, shoot NO3 concentration was not affected by substrate type, while yield and total shoot N and NO3 content were greater when plants were grown in peat than in the mixed substrate or the coir. Leaf chlorophyll meter readings provided a good indication of the amount of N in the plants and increased linearly with total shoot N.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Impact of low temperature storage on active and storage forms of folate in choy sum (Brassica rapa subsp parachinensis)

Choy sum (Brassica rapa subsp. parachinensis) is a dark green leafy vegetable that contains high folate (vitamin B9) levels comparable to spinach. Folate is essential for the maintenance of human health and is obtained solely through dietary means. Analysis of the edible portion of choy sum by both microbiological assay and LC-MS/MS indicated that total folate activity remained significantly unchanged over 3 weeks storage at 4 degrees C. Inedible fractions consisted primarily of outer leaves, which showed signs of rotting after 14d, and a combination of rotting and yellowing after 21 d, contributing to 20% and 40% of product removal, respectively. Following deconjugation of the folate present in choy sum to monoglutamate and diglutamate derivatives, the principal forms (vitamers) of folate detected in choy sum were 5-methyltetrahydrofolate and 5-formyl tetrahydrofolate, followed by tetrahydrofolate (THF), 5,10-methenyl-THF, and 10-formyl folic acid. During storage, a significant decline in 5-formyl-THF was observed, with a slight but not significant increase in the combined 5-methyl-THF derivatives. The decline in 5-formyl-THF in relation to the other folate vitamers present may indicate that 5-formyl-THF is being utilised as a folate storage reserve, being interconverted to more metabolically active forms of folate, such as 5-methyl-THF. Although folate vitamer profile changed over the storage period, total folate activity did not significantly change. From a human nutritional perspective this is important, as while particular folate vitamers (e.g. 5-methyl-THF) are necessary for maintaining vital aspects of plant metabolism, it is less important to the human diet, as humans can absorb and interconvert multiple forms of folate. The current trial indicates that it is possible to store choy sum for up to 3 weeks at 4 degrees C without significantly affecting total folate concentration of the edible portion. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca2+ had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of a- and b-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of a-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by b-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.

Nucleotide sequence of a cucumber chloroplast proline tRNA

The nucleotide sequence of a proline tRNA (anticodon UGG) from cucumber chloroplasts has been determined. The sequence is: pAAGGAUGUAGCGCAGCUUCADAGCGCAΨUUGUUUUGGNΨFACAAAAUm7GUCACGGGTΨCAAAUCCUGUCAUCCUUACCAOH. It shows 93% homology with spinach chloroplast tRNAPro (UGG) and 72% homology with bean mitochondrial tRNA Pro (UGG), the other two known plant organellar tRNAsPro.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light.

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA-treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had no detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These data indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA- treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had not detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These date indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

The conversion of 3-hydroxyanthranilic acid to cinnabarinic acid by the nuclear fraction of rat liver

Although several authors have implicated 3-hydroxyanthranilic acid (3-OHA) as an intermediate in tryptophaniacin pathway in animals (Kaplan, 1961), alternative pathways of metabolism of this compound have not been fully explored. Madhusudanan Nair obtained an enzyme from spinach leaves which could convert 3-OHA to cinnabarinic acid (private communication). Viollier and Süllmann (1950) reported the conversion of 3-OHA to an unidentified red compound by rat liver homogenates. The present investigation describes the identification of this product as cinnabarinic acid (2-amino-3-H-isophenoxazine-3-one-1,9-dicarboxylic acid). Cinnabarinic acid is known to occur in nature along with cinnabarin is olated from the fungus Polystictus sanguineus (Gripenberg et al., 1957; Gripenberg, 1958).

Depletion of the heaviest stable N isotope is associated with NH4 +/NH3 toxicity in NH4 +-fed plants

Background: In plants, nitrate (NO(3)(-)) nutrition gives rise to a natural N isotopic signature (delta(15)N), which correlates with the delta(15)N of the N source. However, little is known about the relationship between the delta(15)N of the N source and the (14)N/(15)N fractionation in plants under ammonium (NH(4)(+)) nutrition. When NH(4)(+) is the major N source, the two forms, NH(4)(+) and NH(3), are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH(3) (g) and NH(4)(+)(aq) which drives to a different delta(15)N. Nine plant species with different NH(4)(+)-sensitivities were cultured hydroponically with NO(3)(-) or NH(4)(+) as the sole N sources, and plant growth and delta(15)N were determined. Short-term NH(4)(+)/NH(3) uptake experiments at pH 6.0 and 9.0 (which favours NH(3) form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH(4)(+) and NH(3). -- Results: Several NO(3)(-)-fed plants were consistently enriched in (15)N, whereas plants under NH(4)(+) nutrition were depleted of (15)N. It was shown that more sensitive plants to NH(4)(+) toxicity were the most depleted in (15)N. In parallel, N-deficient pea and spinach plants fed with (15)NH(4)(+) showed an increased level of NH(3) uptake at alkaline pH that was related to the (15)N depletion of the plant. Tolerant to NH(4)(+) pea plants or sensitive spinach plants showed similar trend on (15)N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO(3) as control discarded that the differences observed arise from pH detrimental effects. -- Conclusions: This article proposes that the negative values of delta(15)N in NH(4)(+)-fed plants are originated from NH(3) uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH(4)(+)/NH(3) toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH(4)(+) may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.

菠菜硬脂酰ACP去饱和酶基因的克隆及其抗寒性转基因烟草的获得

低温是限制植物分布和生物产量的一个重要环境因子。低温的危害也是农业生产上经常遭受的主要自然灾害之一。改良作物的抗寒性是植物科学研究的一个重要课题。植物基因工程的兴起为此目的提供了有力的手段。 大量的研究证明，低温对植物的伤害，首先是使生物膜发生相变和相分离。因此，保持低温下生物膜功能性的液晶态是抗寒的重要机制。研究表明，生物膜这种具流动性的功能态的保持，是与其组成上的膜脂脂肪酸的不饱和度成正相关的。 已有几个关于通过提高膜脂脂肪酸不饱和度的基因操作而增加植物抗寒性的报道。在众多的植物脂肪酸去饱和酶中，硬脂酰ACP去饱和酶(SAD)是最为关键的酶之一。它催化脂肪酸的第一步去饱和反应：18：0-18：l¨。多不饱和脂肪酸是在1 8：1 中由其他去饱和酶再加入双键而生成的。因此，SAD的活性水平是决定植物膜脂不池和度的一个关键因素。 本研究以酸酚法提取的菠菜总RNA为材料，采用反转录一PCR的方法，克隆得到SAD基因，经定向缺失法获得一套该基因的缺失突变体后,用DNA 自动测序仪和双脱氧链终止法测序。获得的SAD基因序列与Nishlda(1992)等发表的菠菜sADcDNA核苷酸序列比较。两者的编码SAD的ORF都为ll97bp，核苷酸差异仅为8bp。但令人惊奇的是我们克隆到的基因，其5‘端上游还存在-个小的ORF，长30bp．编码10个氨基酸。其他报道的SAD基因中都没有这个ORF。 将克隆到的SAD基因构建成两个植物双元表达载体：正义的pB112-13和反义的pB112-6。用叶盘法转化烟草。DNA点杂交和Southern杂交筛选出转基因植株。抗寒性测定表明：当植株置于6'C40小时，转基因植株和对照的相对电导率比较一致，无明显改变;而在88小时，pB121-6转化植株和对照的相对电导率明显升高，以pB1121-6植株升高更多，但pB1121-13转化植株的相对电导率始终保持在较低水平。短暂冰冻处理（-20'C，4O分钟）后置室温下4天，pBl121-6转化烟草总叶绿素含量损失最多，对照次之，而pB1121-13转化烟草中多数植株总叶绿素损伤量都低于其他两种烟草。从这两个抗寒性测定实验，可判定pBl l 2 l—I 3烟株最抗寒，对照次之，而pB1121-6烟株最不抗寒。 由于pB1121-13是增强转基因烟草中SAD活性，而pB1121-6是削弱SAD基因的表达，因此．本研究首次证明通过SAD的基因工程可以改变植物的抗寒性。

菠菜甜菜碱醛脱氢酶的研究

本实验以菠菜叶片为材料，分离并纯化了甜菜碱醛脱氢酶（BADH，EC 1.2.1.8），并对其某些性质进行了研究。此外，还提取并纯化了poly (A)+RNA，并对其完整性进行了分析，主要结果如下： 1.菠菜甜菜碱醛脱氢酶存在于60％硫酸铵沉淀部分，70％硫酸铵沉淀部分未检出其活性。用层析法纯化该酶，使纯化倍数达到405.3倍。菠菜甜菜碱醛脱氢酶有两个同工酶。 2.菠菜甜菜碱醛脱氢酶主要定位于胞液中。其中，在过氧化物酶体及微粒体中有一定量活性存在，但在叶绿体中未检出其活性。 3.菠菜甜菜碱醛脱氢酶活性有较广的pH值范围，其最适pH范围为9.5左右。该酶以NAD作为特异性辅酶，其Km值为8.0×10-6M, Vmax为0.143nmol/min。该酶以甜菜碱醛作为特异性底物，其Km值为1.82×10-4M，Vmax为0.182nmol/min。该酶活性为0.125～1M的NaCl、KCl和脯氨酸所抑制，但0.125～1M的蔗糖及甜菜碱对其活性没有影响。 4.PCMB和Mersalyl抑制该酶的活性，DTT可逐步恢复被抑制的活性。稀土元素LaCl3对该酶活性没有影响，但CeCl则使其完全失活。另外，Mn2+和Mo6+离子对其活性没有影响，Mg2+离子可增加其活性。 5.菠菜叶片中有菠菜甜菜碱醛脱氢酶的抑制因子存在，该因子可能是一种小分子化合物。 6.应用酚-氯仿方法分离了菠菜叶片的poly (A)+RNA，并在-80℃低温下长期贮存。

A record production from an integrated farming system utilising sewage enriched water

The results of experiments conducted on a pond dyke (655m²) in the Wastewater Aquaculture Division of the Central Institute of Freshwater Aquaculture, Rahara, during 1992-93 for maximising production through optimum utilisation of resources are communicated. Round the year intensive cultivation of okra (Abelmoschus esculentus), amaranth (Amaranthus gangeticus and A. viridus), water-bind weed (Ipomea aquatica), Indian spinach (Basella rubra), radish (Raphanus sativum), amaranth (Amaranthus viridis), cauliflower (Brassica oleracia var. votrytis), cabbage (Brassica oleracia var. capitota) and papaya (Carica papaya) was undertaken using the treated sewage water from fish ponds for irrigation. The pond dyke yielded 5,626.5 kg vegetable which worked out to 85.9 tons per ha per year. Multiple cropping with these vegetables excluding papaya on a 460 m² dyke recorded a production of 4,926.5 kg at the rate of 107.1t per ha/yr. An improved yearly net return of about 35% over investment could be achieved through the selection of highly productive and pest resistant vegetable crops of longer duration for integration into the system. Introduction of this type of integrated farming would enhance the overall productivity and returns from farming.