OsLIC1基因参与水稻株型调控的功能研究

CCCH型锌指蛋白是进化上比较保守的一类锌指蛋白家族，其典型的氨基酸的基序为C-X7-8-C-X5-C-X3-H，其中X为任意氨基酸，这类锌指基序一般以重复的双拷贝形式存在。本论文克隆并鉴定了一个全新的、只含有一个CCCH型锌指基序的基因，利用反义RNA策略研究该基因功能，结果发现该基因的反义转基因植株表现出叶夹角增大的表型，因此我们将该基因命名为OsLIC1（Oryza sativa Lamina Increased Leaf Angle Control 1）。生物信息学分析发现该基因定位于水稻6号染色体近端粒的一端，位于BAC克隆AP004324中。OsLIC1与通常的CCCH型锌指蛋白含有多个重复的CCCH锌指基序不同，它只含有一个CCCH型锌指基序。除了CCCH锌指结构域以外，该蛋白在靠近C-端的位置还有一段丝氨酸（Ser）富集的区域，在此区域之前，还有一个在真核生物中相对保守的，以EELR为核心基序的结构域。采用基因枪将含OsLIC1-GFP融合构建的瞬时表达载体轰击入洋葱内表皮细胞，激光共聚焦显微镜观察发现OsLIC1-GFP可以定位到细胞核中。利用酵母转录激活系统发现以EELR为核心基序的结构域具有转录激活的功能。体外核酸结合活性分析显示OsLIC1蛋白可以结合双链DNA，这些结果证明OsLIC1是一个转录因子，这也是在植物中首次发现CCCH型锌指蛋白可以作为转录因子的方式调节基因的表达。 用玉米泛素启动子（Maize Ubiquitin promoter）驱动OsLIC1基因的反义表达载体转化水稻，获得的内源OsLIC1基因表达量下降的转基因植株表现出三个明显的表型：转基因植株的叶夹角增大;转基因的株高低于对照以及转基因植株的穗粒数减少。扫描电镜观察发现转基因植株叶夹角增大是由于近轴面细胞排列发生了改变以及维管束发育受阻引起的。转基因植株的Southern Blot和RT-PCR分析，结合Western Blot分析证明了转基因植株的表型与转基因事件之间的直接联系，并证明了转基因植株中内源OsLIC1在蛋白水平的确受到了抑制。采用RT-PCR技术、Promoter：：GUS和RNA in situ杂交三种方法相结合研究OsLIC1基因的表达模式，结果表明OsLIC1基因主要在叶颈、节以及分蘖原基中表达，这与转基因植株的表型相吻合，进一步证明了转基因植株的表型与基因功能之间的关系。Affymetrix 水稻全基因组芯片分析结果显示许多受油菜素内酯诱导表达的基因在转基因植株的叶颈材料中表达量上调，RT-PCR进一步验证了这一结果。由于在转基因植株中出现的叶夹角增大的表型和水稻油菜素内酯的作用相似，而基因芯片的结果又从分子水平提供了证据和线索。进一步采用RT-PCR和Promoter：：GUS相结合的方法研究OsLIC1基因对油菜素内酯的响应，结果发现OsLIC1基因可以被油菜素内酯诱导表达。而且，OsLIC1基因的反义转基因植株与野生型相比，表现出对油菜素内酯信号更敏感的响应。根据以上结果，推测OsLIC1可能是水稻油菜素内酯信号转导途径的负调控因子。水稻油菜素内酯合成和信号转导的突变体d2-1和d61-1具有直立的叶片的特征。用反义OsLIC1转基因植株以及野生型水稻与d2-1和d61-1突变体分别进行遗传杂交，结果发现在反义OsLIC1转基因植株与d2-1和d61-1突变体的杂交F1代中，都表现出叶夹角增大的表型。但是，在F2代中，在d2-1和d61-1纯合背景下，分别表现出叶夹角增大和叶片直立的表型，说明OsLIC1上位于d2-1，而d61-1则上位于OsLIC1。这一结果进一步证明了OsLIC1是通过参与水稻油菜素内酯信号调节而发挥对水稻叶夹角的调控作用。

Anatomical changes induced by selection for plant architecture in maize (Zea mays L.)

The original brachytic population 'Dent Single Cross Composite' (DSCC-br2br2) and a selection-derived sub-population with modified plant architecture (DSCC-br2br2-Lg3Lg3, selected for erect leaves), were evaluated for the following characteristics number of vascular bundles of greater and smaller size, total vascular tissue area (phloem and xylem), sustaining tissue area (vascular tissue plus sclerenchyma), phloem and sclerenchyma areas in apical, medial and basal portions from midclub and in apical and basal sheath regions (from second leaf above and first below ear insertion). These variables had different values for the five different sections studied in each leaf and these differences did not have the same pattern in the two DSCC populations (brachytic and with modified architecture). Selection for architectural modification caused some indirect foliar anatomical modifications. With the exception of the phloem and the vascular tissue areas in apical leaf and sheath base regions, the modified plant architecture population showed smaller values of sustaining tissue area, sclerenchyma area, vascular tissue area and number of smaller vascular bundles than the original one. In the ligule region the modified maize leaves had smaller vascular and sustaining tissue areas, reducing transportation area, which could reduce gram yield.

Índice de clorofila em plantas de arroz de terras altas submetidas a estresse por alumínio e aplicação de silício

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantification of the effects of architectural traits on dry mass production and light interception of tomato canopy under different temperature regimes using a dynamic functional-structural plant model

There is increasing interest in evaluating the environmental effects on crop architectural traits and yield improvement. However, crop models describing the dynamic changes in canopy structure with environmental conditions and the complex interactions between canopy structure, light interception, and dry mass production are only gradually emerging. Using tomato (Solanum lycopersicum L.) as a model crop, a dynamic functional-structural plant model (FSPM) was constructed, parameterized, and evaluated to analyse the effects of temperature on architectural traits, which strongly influence canopy light interception and shoot dry mass. The FSPM predicted the organ growth, organ size, and shoot dry mass over time with high accuracy (>85%). Analyses of this FSPM showed that, in comparison with the reference canopy, shoot dry mass may be affected by leaf angle by as much as 20%, leaf curvature by up to 7%, the leaf length: width ratio by up to 5%, internode length by up to 9%, and curvature ratios and leaf arrangement by up to 6%. Tomato canopies at low temperature had higher canopy density and were more clumped due to higher leaf area and shorter internodes. Interestingly, dry mass production and light interception of the clumped canopy were more sensitive to changes in architectural traits. The complex interactions between architectural traits, canopy light interception, dry mass production, and environmental conditions can be studied by the dynamic FSPM, which may serve as a tool for designing a canopy structure which is 'ideal' in a given environment.

Simulating droplet motion on virtual leaf surfaces

A curvilinear thin film model is used to simulate the motion of droplets on a virtual leaf surface, with a view to better understand the retention of agricultural sprays on plants. The governing model, adapted from Roy et al. (2002 J. Fluid Mech. 454, 235–261) with the addition of a disjoining pressure term, describes the gravity- and curvature driven flow of a small droplet on a complex substrate: a cotton leaf reconstructed from digitized scan data. Coalescence is the key mechanism behind spray coating of foliage, and our simulations demonstrate that various experimentally observed coalescence behaviours can be reproduced qualitatively. By varying the contact angle over the domain, we also demonstrate that the presence of a chemical defect can act as an obstacle to the droplet’s path, causing break-up. In simulations on the virtual leaf, it is found that the movement of a typical spray size droplet is driven almost exclusively by substrate curvature gradients. It is not until droplet mass is sufficiently increased via coalescence that gravity becomes the dominating force.

QTL for nodal root angle in sorghum (Sorghum bicolor L. Moench) co-locate with QTL for traits associated with drought adaptation.

Nodal root angle in sorghum influences vertical and horizontal root distribution in the soil profile and is thus relevant to drought adaptation. In this study, we report for the first time on the mapping of four QTL for nodal root angle (qRA) in sorghum, in addition to three QTL for root dry weight, two for shoot dry weight, and three for plant leaf area. Phenotyping was done at the six leaf stage for a mapping population (n = 141) developed by crossing two inbred sorghum lines with contrasting root angle. Nodal root angle QTL explained 58.2% of the phenotypic variance and were validated across a range of diverse inbred lines. Three of the four nodal root angle QTL showed homology to previously identified root angle QTL in rice and maize, whereas all four QTL co-located with previously identified QTL for stay-green in sorghum. A putative association between nodal root angle QTL and grain yield was identified through single marker analysis on field testing data from a subset of the mapping population grown in hybrid combination with three different tester lines. Furthermore, a putative association between nodal root angle QTL and stay-green was identified using data sets from selected sorghum nested association mapping populations segregating for root angle. The identification of nodal root angle QTL presents new opportunities for improving drought adaptation mechanisms via molecular breeding to manipulate a trait for which selection has previously been very difficult.

Remote sensing of leaf area index: enhanced retrieval from close-range and remotely sensed optical observations

A wide range of models used in agriculture, ecology, carbon cycling, climate and other related studies require information on the amount of leaf material present in a given environment to correctly represent radiation, heat, momentum, water, and various gas exchanges with the overlying atmosphere or the underlying soil. Leaf area index (LAI) thus often features as a critical land surface variable in parameterisations of global and regional climate models, e.g., radiation uptake, precipitation interception, energy conversion, gas exchange and momentum, as all areas are substantially determined by the vegetation surface. Optical wavelengths of remote sensing are the common electromagnetic regions used for LAI estimations and generally for vegetation studies. The main purpose of this dissertation was to enhance the determination of LAI using close-range remote sensing (hemispherical photography), airborne remote sensing (high resolution colour and colour infrared imagery), and satellite remote sensing (high resolution SPOT 5 HRG imagery) optical observations. The commonly used light extinction models are applied at all levels of optical observations. For the sake of comparative analysis, LAI was further determined using statistical relationships between spectral vegetation index (SVI) and ground based LAI. The study areas of this dissertation focus on two regions, one located in Taita Hills, South-East Kenya characterised by tropical cloud forest and exotic plantations, and the other in Gatineau Park, Southern Quebec, Canada dominated by temperate hardwood forest. The sampling procedure of sky map of gap fraction and size from hemispherical photographs was proven to be one of the most crucial steps in the accurate determination of LAI. LAI and clumping index estimates were significantly affected by the variation of the size of sky segments for given zenith angle ranges. On sloping ground, gap fraction and size distributions present strong upslope/downslope asymmetry of foliage elements, and thus the correction and the sensitivity analysis for both LAI and clumping index computations were demonstrated. Several SVIs can be used for LAI mapping using empirical regression analysis provided that the sensitivities of SVIs at varying ranges of LAI are large enough. Large scale LAI inversion algorithms were demonstrated and were proven to be a considerably efficient alternative approach for LAI mapping. LAI can be estimated nonparametrically from the information contained solely in the remotely sensed dataset given that the upper-end (saturated SVI) value is accurately determined. However, further study is still required to devise a methodology as well as instrumentation to retrieve on-ground green leaf area index . Subsequently, the large scale LAI inversion algorithms presented in this work can be precisely validated. Finally, based on literature review and this dissertation, potential future research prospects and directions were recommended.

Effect of nozzle angle and air-jet parameters in air-assisted sprayer on biological effect of soybean asian rust chemical protection

Aiming at improving the efficiency control of Phakopsora pachyrhizi, this research evaluated different application techniques, using spray deposits and yield parameters of soybean crop. Two experiments were carried out in the experimental area of FCA/UNESP - Botucatu, SP, Brazil, in the soybean crop, Conquista variety, in the 2006/2007 season. The first experiment was arranged in random blocks with eight treatments and four replications. The treatments were conducted in factorial arrangement 4×2 (four air levels 0, 9, 11 and 29 km/h combined at two nozzle angles 0 and 30°) using AXI 110015 nozzles. Ten plants on each plot were selected for sampling spray deposits. Artificial targets were fixed on plants, two in the top and another two in the bottom part of plants (abaxial and adaxial leaf surface each one). For deposit evaluations, a cupric tracer was used and the amount of deposits was determined by a spectrophotometer. The second experiment was carried out in the same place and the treatments were of the same arrangement as the previous experiment, including control treatment (untreated plants). The spraying with triazole fungicide was realized in R2 and R5.2 growth stages of soybean with 142 l/ha spray volume. The nozzle angled of 30° combined with maximum air speed promoted the highest spray deposits on the soybean crop and influenced positively the control of the soybean Asian rust as well in the productivity of this crop.

Microsurgical and laser ablation analysis of leaf positioning and dorsoventral patterning in tomato

Leaves are arranged according to regular patterns, a phenomenon referred to as phyllotaxis. Important determinants of phyllotaxis are the divergence angle between successive leaves, and the size of the leaves relative to the shoot axis. Young leaf primordia are thought to provide positional information to the meristem, thereby influencing the positioning of new primordia and hence the divergence angle. On the contrary, the meristem signals to the primordia to establish their dorsoventral polarity, which is a prerequisite for the formation of a leaf blade. These concepts originate from classical microsurgical studies carried out between the 1920s and the 1970s. Even though these techniques have been abandoned in favor of genetic analysis, the resulting insights remain a cornerstone of plant developmental biology. Here, we employ new microsurgical techniques to reassess and extend the classical studies on phyllotaxis and leaf polarity. Previous experiments have indicated that the isolation of an incipient primordium by a tangential incision caused a change of divergence angle between the two subsequent primordia, indicating that pre-existing primordia influence further phyllotaxis. Here.. we repeat these experiments and compare them with the results of laser ablation of incipient primordia. Furthermore. we explore to what extent the different pre-existing primordia influence the size and position of new organs. and hence phyllotaxis. We propose that the two youngest primordia (P-1 and P-2) are sufficient for the approximate positioning of the incipient primordium (I-1), and therefore for the perpetuation of the generative spiral, whereas the direct contact neighbours of I-1 (P-2 and P-3) control its delimitation and hence its exact size and position. Finally. we report L I specific cell ablation experiments suggesting that the meristem L-1 layer is essential for the dorsoventral patterning of leaf primordia.

Interactions between Senescence and Leaf Orientation Determine in Situ Patterns of Photosynthesis and Photoinhibition in Field-Grown Rice1

Photosynthesis and photoinhibition in field-grown rice (Oryza sativa L.) were examined in relation to leaf age and orientation. Two varieties (IR72 and IR65598-112-2 [BSI206]) were grown in the field in the Philippines during the dry season under highly irrigated, well-fertilized conditions. Flag leaves were examined 60 and 100 d after transplanting. Because of the upright nature of 60-d-old rice leaves, patterns of photosynthesis were determined by solar movements: light falling on the exposed surface in the morning, a low incident angle of irradiance at midday, and light striking the opposite side of the leaf blade in the afternoon. There was an early morning burst of CO2 assimilation and high levels of saturation of photosystem II electron transfer as incident irradiance reached a maximum level. However, by midday the photochemical efficiency increased again almost to maximum. Leaves that were 100 d old possessed a more horizontal orientation and were found to suffer greater levels of photoinhibition than younger leaves, and this was accompanied by increases in the de-epoxidation state of the xanthophyll cycle. Older leaves had significantly lower chlorophyll content but only slightly diminished photosynthesis capacity.