Vol 64, No 4 (2017)

Reduction in the area occupied with Bt-crops caused by their cost inefficiency largely depends on the outbreaks of secondary pests. Among them, a highly important place belongs to the representatives of the order Hemiptera. These phytophages are insensitive to Cry-proteins of genetically modified (GM) insecticidal crops engineered to control the main pests from the orders Lepidoptera and Coleoptera with chewing mouthparts. In contrast to chewing, evolutionary more advanced piercing-sucking mouthparts of Hemiptera phytophages do not initiate in plants the state of induced defense associated with an elevated content of terpenes. When the main pest is absent, a reduced content of terpenes in Bt-crops makes them more attractive to hemipterans than conventional cultivars. In the absence of the main pest there’s no competition for hemipterous secondary pests. In aggregate, these two factors bring about outbreaks of Hemiptera in Bt-crops.

The main objective of the present study is to test the effect of short preexposure of lentil (Lens culinaris Medik.) plants to low salt concentration on shoot growth, oxidative stress and activity of antioxidant enzymes under high salt stress. To fulfill this objective, lentil plants were pretreated with 10 mM NaCl for 3 days and then they were exposed to high salt concentration of 300 mM for 7 days. After that, shoot growth was evaluated in terms of shoot length, fresh and dry weight. Biochemical changes in terms of oxidative stress and activity of antioxidant enzymes were also assessed in lentil plants. The shoot growth of lentil plants preexposed to low salt concentration was significantly enhanced under high salt stress, whereas it was severely retarded in lentil plants that were directly exposed to high salt stress. Moreover, lipid peroxidation and the accumulation of hydrogen peroxide were highly reduced in the shoot of lentil plants preexposed to low salt concentration. The activity of antioxidant enzymes (catalase and superoxide dismutase) was significantly higher in the shoot of lentil plants preexposed to low salt concentration than those directly exposed to high salt concentration. Overall, the results revealed an enhanced salt tolerance in lentil plants after short exposure to low salt concentration.

Quantitative real-time polymerase chain reaction (qRT-PCR) has been applied to determine the expression levels of Dendrobium catenatum genes. However, so far, the suitable reference genes in the qRT-PCR analysis have not been established in this case. In this study, seven housekeeping genes of D. catenatum, i.e., those of actin 1 (ACT1), cyclophilin (CYP), eukaryotic initiation factor (eIF), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), α-tubulin (TUA), β-tubulin (TUB) and ubiquitin-conjugating enzyme (UBC), were selected as the candidate references for the qRT-PCR analysis of D. catenatum genes. Based on cDNA library, the cDNAs of the seven genes were cloned by reverse transcription (RT-PCR), and the transcription levels of these genes in different organs and tissues of D. catenatum were assessed by qRT-PCR; the expression stability of the genes was determined with BestKeeper, GeNorm and NormFinder programs, respectively. The open reading frames (ORFs) of the cDNAs of the seven genes were 1134, 522, 342, 1020, 1356, 1344 and 459 bp long, respectively. The corresponding amino acid sequences shared 87–100% identities with the prototype proteins registered in the GenBank, and held some specific conserved motifs required for the biological functions of these proteins. According to their expression stability, DcUBC, DcCYP and DcGAPDH would be preferentially selected as the reference genes for the qRT-PCR analysis of D. catenatum genes. Based on their expression levels, the most suitable reference ones were DcCYP, DcGAPDH and DcUBC in descending order.

The present study assessed the response of pea plants exposed to herbicide induced oxidative stress in the plants present in agriculture field. We analysed the effect of exogenous nitric oxide (NO) regulated chlorophyll and protein content, nitrate reductase enzyme activity and antioxidant enzyme activity in herbicidetreated green pea (Pisum sativum L.). Glyphosate (0.25 mM) treatment alone or in combination with 250 μM sodium nitroprusside (SNP, 250 μM with glyphosate) was given to pea and we observed the changes in biophysical and biochemical parameters. During oxidative stress ion leakage is the first step of cellular damage. Supplementation of SNP with glyphosate significantly reduced ion leakage and moderately reduced H2O2 and malondialdehyde (MDA) content. SNP also increased chlorophyll content and antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (POD) activity as compared to herbicide treatment alone. The present result suggests that NO protects pea plants from damage caused by glyphosate.

We studied the temporal sequence of changes in the photosynthetic CO₂/H₂O gas exchange intensity, as well as leaf water status, contents of soluble carbohydrates, starch, proline, pigments, and MDA, in maize seedlings (Zea mays L., cv. Luchistaya) under adaptation to increasing water deficit. The duration of drought was 2, 3, 5, and 6 days. Withholding water from maize plants caused gradual increase in the intensity of water deficit: from mild (2 or 3 days) to moderate (5 days) and nearly severe (6 days) water stress. After 6 days, relative leaf water content decreased by 19.8% as compared to the control. On the second day after the onset of drought, slight reduction in the photosynthetic CO₂/H₂O gas exchange intensity of the treated plants was observed. After 6 days, photosynthesis and transpiration of leaves synchronously reduced almost threefold due to stomatal closure. The progressive soil drought had substantial impact on the carbohydrate metabolism. After 2 days of water deficit, the content of reducing sugars and sucrose increased slightly, whereas after 6 days, it increased ten and four times, respectively. After 2, 3, and 5 days of drought, the starch content declined slightly; however, under severe drought (6 days), it increased by 30% as compared to the control. Simultaneously with the increase in the content of soluble sugars, proline content increased significantly and it was the highest on the sixth day of drought. At all stages of water deficit, the proline content increased more significantly than the content of reducing carbohydrates and sucrose. Under increasing water deficit (5 and 6 days), the content of MDA was found to rise. At the initial drought stage (2 or 3 days) and under severe water deficit (6 days), no significant changes in the pigment content were observed. Thus, at the initial stages of progressive drought, in the leaves of this maize cultivar, a decline in photosynthetic activity proceeded simultaneously with accumulation of reducing sugars, sucrose, and proline. The results obtained showed that, at the first stages of adaptation of maize seedlings to drought, the changes in carbohydrate and proline metabolism have been observed, which have increased upon further plant dehydration.

Salvia miltiorrhiza Bunge produces salvianolic acid, a water-soluble phenolic acid that is widely used in treating cardiovascular disease. The objective of this study is to employ AtPAP1 (Production of Anthocyanin Pigmentation) and SmCCR1 (cinnamoyl CoA reductase) for improving the phenolic acids of S. miltiorrhiza. First, results showed that AtPAP1 significantly increased the promoter activities of SmCCR1 by transient transformation of tobacco. To enrich the precursors available for phenolic acid biosynthesis, we present a strategy of combinational genetic manipulation by Agrobacterium tumefaciens-mediated gene transfer that simultaneously over-expressed AtPAP1 and down-regulated SmCCR1. Compared with the control, the amount of lignin was significantly reduced and its composition was changed in transgenic plants. While, the main phenolic acids, including salvianolic acid B and rosmarinic acid, were induced 2.0- and 1.8-fold compared with control lines in two-month-old plants. Meanwhile, the contents of total phenolics and total flavonoids were significantly improved in transformed plants. Expression of related genes throughout the phenylpropanoid pathways were altered, following the same trends as shown for their products, i.e., phenolic acids accumulation and lignin reduction. These results demonstrate that such methods for genetic modification, which increase the expression of transcription factors while suppressing that of branch genes, are of great value when genetically engineering plants for other secondary products.

Rubus idaeus L. is of great economic value. Some varieties of Rubus idaeus have a unique feature of spontaneous rooting from the stem apex. To determine whether DNA methylation is associated with the spontaneous rooting process, variations in the methylation at stem apex during four root developmental stages were investigated, using the methylation-sensitive amplification polymorphism (MSAP) technique and the bisulfite sequencing analysis (BSA). The results showed that the DNA methylation levels and patterns were significantly different between the four developmental stages. A total of 824 CCGG amplified sites were detected by MSAP. MSAP screening revealed that the level of DNA methylation at stages I to IV was 34.95, 36.04, 36.29, and 37.50%, respectively. The number of methylated sites and their methylation levels tended to decrease at stages III and IV (root differentiation and elongation) compared with those at stage I (stem elongation). After cloning and sequencing of the 16 polymorphic differentially methylated DNA fragments, BLAST search results indicated that they might be involved in differentiation of the lateral root primordium, plant defense, signal transduction, and energy metabolism. Results of the qRT-PCR and BSA analyses confirmed that methylation of some key genes was closely associated with their expression at the different developmental stages. These findings could be useful for future studies on the potential role of DNA methylation in spontaneous rooting from the stem apex, implying its importance in rooting regulation and rapid expansion of raspberry populations.

Seeds of Bunium persicum (Boiss.) B. Fedtsch. have complex physiological dormancy that can be released by 15 weeks stratification. The present study revealed that cold stratification enhanced content of H₂O₂, O^2-· and application of GA₃ and ROS donors (Fenton reagent, H₂O₂, methylviologen and menadione) did not affect or only slightly promoted the germination of non-stratified, fully dormant seeds. Dormancy was markedly decreased by ROS-generating reagents, GA₃ and fluridone (an inhibitor of ABA biosynthesis) and was enhanced by ROS-decreasing compounds (DMTU, Tiron, SB and DPI), diniconazole (Dinc, an inhibitor of ABA catabolism) and paclobutrazol (PAC, an inhibitor of GA biosynthesis) when dormancy was partially removed by cold stratification. The response to these compounds reduced with increasing time of stratification. ABA inhibited germination by repressing of NADPH oxidase activity and ROS accumulation and conversely, GA triggered germination by promoting an increase of NADPH oxidase activity and ROS levels. Data in this study, for the first time suggest releasing deep complex physiological dormancy by cold stratification is associated with interplay between ROS and ABA/GA.

Millet colour is an important index to evaluate the quality of dehulled foxtail millet (Setaria italica (L.) Beauv.). Most varieties are yellow, due to the accumulation of carotenoids. However, there are some foxtail millet germplasms producing dark green millet. To elucidate the molecular mechanism of the chlorophyll retention phenotype, Daqinggu with green millet colour and Jingu 21 with yellow millet colour were selected as research material in this study. The total carotenoid level in dehulled millet of Daqinggu was about 0.024 mg/g, and 0.038 mg/g in Jingu 21. The transcript levels of carotenoid structural genes were investigated at three stages of grain development in both millet varieties. The expression levels of carotenoid biosysnthesis-related genes SiPSY3, SiPDS, SiZ-ISO, SiLCYB and SiCYP97C were significantly higher in Daqinggu than in Jingu 21, which was not consistent with the difference in the carotenoid levels between these two varieties. Interestingly, SiSGR, a homologue to the STAY-GREEN gene in Arabidopsis, tomato, and rice, was constitutively down-regulated during maturation in Daqinggu. In addition, the total chlorophyll content was consistently higher in Daqinggu than in Jingu21 during grain maturation. These evidences suggest that SiSGR is a key gene in regulating chlorophyll retention for dark green foxtail millet.

Life strategy of plants depends on successful seed germination in the available environment, and sufficient soil water is the most important external factor. Taking into account a broad spectrum of roles played by water in seed viability and its maintenance during germination, the review embraces early germination events in seeds different in their water status. Two seed types are compared, namely orthodox and recalcitrant seeds, in terms of water content in the embryonic axes, vacuole biogenesis, and participation of water channels in membrane water transport. Mature orthodox seeds desiccate to low water content and remain viable during storage, whereas mature recalcitrant seeds are shed while well hydrated but die during desiccation and cannot be stored. In orthodox Vicia faba minor air-dry seeds remaining viable at 8–10% water content in embryonic axes, the vacuoles in hypocotyl are preserved as protein storage vacuoles, then restored to vacuoles in imbibing seeds in the course of protein mobilization. However, in newly produced meristematic root cells, the vacuoles are formed de novo from provacuoles. In recalcitrant Aesculus hippocastanum seeds, embryonic axes have a water content of 63–64% at shedding and they lack protein storage vacuoles but preserve vacuoles preformed in maturing seeds. Independent of the vacuolar biogenetic patterns, their further trend is similar; they expand and fuse, thus producing an osmotic compartment, which precedes and becomes an obligatory step for the initiation of cell elongation. Prior to this, water moves in imbibing seeds through the membranes by diffusion, although the aquaporins forming water channels are present. In both seed types, water channels are opened and actively participate in water transport only after growth initiation. Aquaporin gene expression and their composition change in broad bean embryonic axes after growth initiation. This is the way how a mass water flow into growing seedling cells is achieved, independent of differences in seed water content and vacuole biogenesis patterns.