Aleksandar Cingel

Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings

The effects of Chenopodium murale root exudates, applied as phytotoxic medias (PMs), were tested on Arabidopsis thaliana and Triticum aestivum. The effects of PMs, where wild-type roots (K), hairy roots derived from roots (R clones) or from cotyledons (C clones) were cultured, were different. K medium suppressed Arabidopsis germination, while other PMs reduced root and leaf elongation and the number of rosette leaves. R media were more phytotoxic than C media. Treatment of Arabidopsis with R8 down-regulated expression of core cell cycle genes: cyclin-dependent kinase (CDK) A1;1, four B-class CDKs, and cyclins CYCA3;1, CYCB2;4, CYCD4;2 and CYCH1 in root and shoot tips. Only CYCD2;1 transcript was elevated in treated shoots, but down-regulated in roots. Wheat Ta-CDC2 and Ta-CYCD2 genes showed the same expression profiles as their Arabidopsis counterparts, CDKA1;1 and CYCD2;1. PMs also caused increase of antioxidative enzyme activities in both plants. Exposure of Arabidopsis to PMs induced one catalase isoform, but repressed another, resulting in no net change of catalase activity. Wheat seedlings treated with PMs had catalase activity significantly elevated in all treatments, particularly in shoots. In both plants, PMs induced the activity of different peroxidase isozymes and total peroxidase activity. Both plants responded to phytotoxic treatments by induction of CuZn-superoxide dismutase. Thus, the phytotoxicity of C. murale root exudates is, at least partially, based on down-regulation of the cell cycle regulators and on generation of oxidative stress in the affected plants. We propose that C. murale root exudates should be considered as means of biological weed control.

Pyramiding rice cystatin OCI and OCII genes in transgenic potato (Solanum tuberosum L.) for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say)

Proteinase inhibitors oryzacystatins I and II (OCI and OCII) have shown potential in controlling pests that utilize cysteine proteinases for protein digestion. In order to achieve an additive effect against Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) digestive proteinases, we combined two individual OC genes in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica. The OC genes were stacked into the potato genome by simultaneous co-transformation with both OCI and OCII genes or by sequential re-transformation of an OCI-expressing transgenic line with an OCII gene. Low constitutive and high wound-induced transcript levels of both OCI and OCII genes, directed by the inducible pin2 promoter, were detected in all doubly transformed lines from all three cultivars. Both recombinant OCs, detected by immunoblot, were in an active conformation as shown by their strong papain inhibitory activity. Although no significant differences in CPB larval mortality were observed when larvae fed on OCI/OCII transformed or control potato foliage, expression of both OCI and OCII in potato plants caused a reduction in CPB adult body weight and in weight of foliage consumed, indicating an additive effect. Overall, these results show that pyramiding of the OCI and OCII genes could be an effective strategy for breeding cultivars exhibiting elevated levels of resistance to CPB.

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

Extraordinary Adaptive Plasticity of Colorado Potato Beetle: “Ten-Striped Spearman” in the Era of Biotechnological Warfare

Expanding from remote areas of Mexico to a worldwide scale, the ten-striped insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), has risen from being an innocuous beetle to a prominent global pest. A diverse life cycle, phenotypic plasticity, adaptation to adverse conditions, and capability to detoxify or tolerate toxins make this insect appear to be virtually “indestructible”. With increasing advances in molecular biology, tools of biotechnological warfare were deployed to combat CPB. In the last three decades, genetically modified potato has created a new challenge for the beetle. After reviewing hundreds of scientific papers dealing with CPB control, it became clear that even biotechnological means of control, if used alone, would not defeat the Colorado potato beetle. This control measure once again appears to be provoking the potato beetle to exhibit its remarkable adaptability. Nonetheless, the potential for adaptation to these techniques has increased our knowledge of this pest and thus opened possibilities for devising more sustainable CPB management programs.

Changes in cytokinin content and altered cytokinin homeostasis in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro

The plant hormones cytokinins (CKs) regulate a number of physiological processes. Their homeostasis is controlled by the rate of de novo synthesis and the rate of catabolism. The aim of this work was to analyze the content of total as well as individual groups of endogenous CKs in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro. Transgenic CKX plants represent a suitable model system for studying physiological and morphological processes controlled by CKs. In this work we clearly demonstrate a significant effect of AtCKX transgenes on CK metabolism in transgenic centaury plants. However, shoots and roots of only one AtCKX1 line and three AtCKX2 lines with a significant reduction of bioactive CKs were obtained. We also show that changes in the CKs metabolism considerably affected endogenous indole-3-acetic acid (IAA) levels in plant tissues. All analyzed transgenic AtCKX centaury lines exhibited decreased amount of endogenous IAA in shoots as well as in roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all analyzed AtCKX centaury transformants.