Automated image analysis for quantification of histochemical detection of reactive oxygen species and necrotic infection symptoms in plant leaves

Abstract

In plants, the accelerated production of reactive oxygen species (ROS) is a common response to different stresses. We developed a computer method of leaf image analysis to automatically quantify the O À 2 and H 2 O 2 staining patterns in leaves. The quantification of histochemically detected ROS is given as a percentage of stained area partitioned according to the staining intensity and hence O À 2 =H 2 O 2 accumulation. Thus, this method expands the routine histochemical detection of ROS in situ. The same procedure can be also used to assess necrotic infection symptoms. In plants infected with necrotizing pathogens leaves can be simultaneously examined for pathogeninduced O À 2 and H 2 O 2 accumulation and infection development. This procedure allows rapid and accurate examination of many leaves and provides an advantage over the routine visual assessment of in situ histochemical detection of ROS and infection symptom development.

Hormetic Responses of Photosystem II in Tomato to Botrytis cinerea

“…In plant cellular metabolism, and especially in chloroplasts, reactive oxygen species (ROS) are continuously produced at basal levels that are incapable to cause damage, as they are being scavenged by different antioxidant mechanisms [ 9 , 10 , 11 , 12 ]. However, under most biotic or abiotic stresses an increased production of ROS occurs that can lead to oxidative stress if it is not scavenged by enzymatic or non-enzymatic antioxidants [ 11 , 12 , 13 , 14 , 15 ]. However, ROS derived from the chloroplasts also play a role in plant resistance against B. cinerea [ 15 ].…”

“…However, under most biotic or abiotic stresses an increased production of ROS occurs that can lead to oxidative stress if it is not scavenged by enzymatic or non-enzymatic antioxidants [ 11 , 12 , 13 , 14 , 15 ]. However, ROS derived from the chloroplasts also play a role in plant resistance against B. cinerea [ 15 ].…”

“…ROS, such as superoxide anion radical (O 2 • − ), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ), produced in chloroplasts play dual roles as they generate oxidative stress and also confer essential biological function as redox signaling in response to biotic and abiotic stress conditions [ 14 , 15 , 16 ]. The role of chloroplast antioxidants, that often have overlying or interrelating functions, is not to totally eliminate ROS, but rather to achieve a suitable balance between production and removal so that to counterpart photosynthetic function, permitting an effective diffusion of signals to the nucleus and adjusting a plethora of physiological functions [ 13 , 16 , 17 ].…”

“…The role of chloroplast antioxidants, that often have overlying or interrelating functions, is not to totally eliminate ROS, but rather to achieve a suitable balance between production and removal so that to counterpart photosynthetic function, permitting an effective diffusion of signals to the nucleus and adjusting a plethora of physiological functions [ 13 , 16 , 17 ]. Leaf image analysis is also used as an objective and accurate alternative method to quantify ROS production at the affected leaf areas, and has proven to be a useful tool in plant science [ 15 ].…”

Spatial referencing of chlorophyll fluorescence images for quantitative assessment of infection propagation in leaves demonstrated on the ice plant: Botrytis cinerea pathosystem

“…Chlorophyll fluorescence provides images map on photosynthetic organs the variations of single fluorescence parameters in false-colour mode. The fluorescence parameters are usually quantified as the average values from identified stress regions, as for other computer-assisted image analyses [6, 7], obtained from manually-defined areas of interest. The performance of photosynthesis is highly heterogeneous over a single leaf surface and between leaves of a plant and the locally-specific changes driven by a stressor can be masked when the entire leaf surface is processed quantitatively.…”