Digital image analysis as a tool to estimate legume contributions in legume-grass swards

Summary: Productivity and forage quality of legume-grass swards are important factors for successful arable farming in both organic and conventional farming systems. For these objectives the botanical composition of the swards is of particular importance, especially, the content of legumes due to their ability to fix airborne nitrogen. As it can vary considerably within a field, a non-destructive detection method while doing other tasks would facilitate a more targeted sward management and could predict the nitrogen supply of the soil for the subsequent crop. This study was undertaken to explore the potential of digital image analysis (DIA) for a non destructive prediction of legume dry matter (DM) contribution of legume-grass mixtures. For this purpose an experiment was conducted in a greenhouse, comprising a sample size of 64 experimental swards such as pure swards of red clover (Trifolium pratense L.), white clover (Trifolium repens L.) and lucerne (Medicago sativa L.) as well as binary mixtures of each legume with perennial ryegrass (Lolium perenne L.). Growth stages ranged from tillering to heading and the proportion of legumes from 0 to 80 %. Based on digital sward images three steps were considered in order to estimate the legume contribution (% of DM): i) The development of a digital image analysis (DIA) procedure in order to estimate legume coverage (% of area). ii) The description of the relationship between legume coverage (% area) and legume contribution (% of DM) derived from digital analysis of legume coverage related to the green area in a digital image. iii) The estimation of the legume DM contribution with the findings of i) and ii). i) In order to evaluate the most suitable approach for the estimation of legume coverage by means of DIA different tools were tested. Morphological operators such as erode and dilate support the differentiation of objects of different shape by shrinking and dilating objects (Soille, 1999). When applied to digital images of legume-grass mixtures thin grass leaves were removed whereas rounder clover leaves were left. After this process legume leaves were identified by threshold segmentation. The segmentation of greyscale images turned out to be not applicable since the segmentation between legumes and bare soil failed. The advanced procedure comprising morphological operators and HSL colour information could determine bare soil areas in young and open swards very accurately. Also legume specific HSL thresholds allowed for precise estimations of legume coverage across a wide range from 11.8 - 72.4 %. Based on this legume specific DIA procedure estimated legume coverage showed good correlations with the measured values across the whole range of sward ages (R2 0.96, SE 4.7 %). A wide range of form parameters (i.e. size, breadth, rectangularity, and circularity of areas) was tested across all sward types, but none did improve prediction accuracy of legume coverage significantly. ii) Using measured reference data of legume coverage and contribution, in a first approach a common relationship based on all three legumes and sward ages of 35, 49 and 63 days was found with R2 0.90. This relationship was improved by a legume-specific approach of only 49- and 63-d old swards (R2 0.94, 0.96 and 0.97 for red clover, white clover, and lucerne, respectively) since differing structural attributes of the legume species influence the relationship between these two parameters. In a second approach biomass was included in the model in order to allow for different structures of swards of different ages. Hence, a model was developed, providing a close look on the relationship between legume coverage in binary legume-ryegrass communities and the legume contribution: At the same level of legume coverage, legume contribution decreased with increased total biomass. This phenomenon may be caused by more non-leguminous biomass covered by legume leaves at high levels of total biomass. Additionally, values of legume contribution and coverage were transformed to the logit-scale in order to avoid problems with heteroscedasticity and negative predictions. The resulting relationships between the measured legume contribution and the calculated legume contribution indicated a high model accuracy for all legume species (R2 0.93, 0.97, 0.98 with SE 4.81, 3.22, 3.07 % of DM for red clover, white clover, and lucerne swards, respectively). The validation of the model by using digital images collected over field grown swards with biomass ranges considering the scope of the model shows, that the model is able to predict legume contribution for most common legume-grass swards (Frame, 1992; Ledgard and Steele, 1992; Loges, 1998). iii) An advanced procedure for the determination of legume DM contribution by DIA is suggested, which comprises the inclusion of morphological operators and HSL colour information in the analysis of images and which applies an advanced function to predict legume DM contribution from legume coverage by considering total sward biomass. Low residuals between measured and calculated values of legume dry matter contribution were found for the separate legume species (R2 0.90, 0.94, 0.93 with SE 5.89, 4.31, 5.52 % of DM for red clover, white clover, and lucerne swards, respectively). The introduced DIA procedure provides a rapid and precise estimation of legume DM contribution for different legume species across a wide range of sward ages. Further research is needed in order to adapt the procedure to field scale, dealing with differing light effects and potentially higher swards. The integration of total biomass into the model for determining legume contribution does not necessarily reduce its applicability in practice as a combined estimation of total biomass and legume coverage by field spectroscopy (Biewer et al. 2009) and DIA, respectively, may allow for an accurate prediction of the legume contribution in legume-grass mixtures.

Not the outside but the inside matters : functional analysis of Capricious and Tartan during Drosophila tracheal morphogenesis

The tubular structures, which transport essential gases, liquids, or cells from one site to another, are shared among various divergent organisms. These highly organized tubular networks include lung, kidney, vasculature and mammary gland in mammals as well as trachea and salivary gland in Drosophila melanogaster. Many questions regarding the tubular morphogenesis cannot be addressed sufficiently by investigating the mammalian organs because their structures are extremely complex and therefore, systematic analyses of genetic and cellular programs guiding the development is not possible. In contrast, the Drosophila tracheal development provides an excellent model system since many molecular markers and powerful tools for genetic manipulations are available. Two mechanisms were shown to be important for the outgrowth of tracheal cells: the FGF signaling pathway and the interaction between the tracheal cells and the surrounding mesodermal cells. The Drosophila FGF ligand encoded by branchless (bnl) is localized in groups of cells near tracheal metameres. The tracheal cells expressing the FGF receptor breathless (btl) respond to these sources of FGF ligand and extend towards them. However, this FGF signaling pathway is not sufficient for the formation of continuous dorsal trunk, the only muticellular tube in tracheal system. Recently, it was found out that single mesodermal cells called bridge-cells are essential for the formation of continuous dorsal trunk as they direct the outgrowth of dorsal trunk cells towards the correct targets. The results in this PhD thesis demonstrate that a cell adhesion molecule Capricious (Caps), which is specifically localized on the surface of bridge-cells, plays an essential role in guiding the outgrowing dorsal trunk cells towards their correct targets. When caps is lacking, some bridge-cells cannot stretch properly towards the adjacent posterior tracheal metameres and thus fail to interconnect the juxtaposing dorsal trunk cells. Consequently, discontinuous dorsal trunks containing interruptions at several positions are formed. On the other hand, when caps is ectopically expressed in the mesodermal cells through a twi-GAL4 driver, these mesodermal cells acquire a guidance function through ectopic caps and misguide the outgrowing dorsal trunk cells in abnormal directions. As a result, disconnected dorsal trunks are formed. These loss- and gain-of-function studies suggest that Caps presumably establishes the cell-to-cell contact between the bridge-cells and the tracheal cells and thereby mediates directly the guidance function of bridge-cells. The most similar protein known to Caps is another cell adhesion molecule called Tartan (Trn). Interestingly, trn is expressed in the mesodermal cells but not in the bridge-cells. When trn is lacking, the outgrowth of not only the dorsal trunks but also the lateral trunks are disrupted. However, in contrast to the ectopic expression of caps, the misexpression of trn does not affect tracheal development. Whereas Trn requires only its extracellular domain to mediate the matrix function, Caps requires both its extracellular and intracellular domains to function as a guidance molecule in the bridge-cells. These observations suggest that Trn functions differently from Caps during tracheal morphogenesis. Presumably, Trn mediates a matrix function of mesodermal cells, which support the tracheal cells to extend efficiently through the surrounding mesodermal tissue. In order to determine which domains dictate the functional specificity of Caps, two hybrid proteins CapsEdTrnId, which contains the Caps extracellular domain and the Trn intracellular domain, and TrnEdCapsId, which consists of the Trn extracellular domain and the Caps intracellular domain, were constructed. Gain of function and rescue experiments with these hybrid proteins suggest on one hand that the extracellular domains of Caps and Trn are functionally redundant and on the other hand that the intracellular domain dictates the functional specificity of Caps. In order to identify putative interactors of Caps, yeast two-hybrid screening was performed. An in vivo interaction assay in yeast suggests that Ras64B interacts specifically with the Caps intracellular domain. In addition, an in vitro binding assay reveals a direct interaction between an inactive form of Ras64B and the Caps intracellular domain. ras64B, which encodes a small GTPase, is expressed in the mesodermal cells concurrently as caps. Finally, a gain-of-function study with the constitutively active Ras64B suggests that Ras64B presumably functions downstream of Caps. All these results suggest consistently that the small GTPase Ras64B binds specifically to the Caps intracellular domain and may thereby mediate the guidance function of Caps.