Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat,drought and their combination in Nicotiana tabacum plants

Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.     

LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in arabidopsis

In plants, light is not only an energy source but also a very important signal that modulates development and differentiation. Here, we report a putative photo-regulatory factor sequence in LKP1 (LOV kelch protein 1). LKP1 cDNA encodes a protein of 610 amino acids and with a molecular weight of 65 905 with an LOV domain and kelch repeats. LOV domains are present in a number of sensor proteins involved in the detection of light, oxygen or voltage. The LKP1 LOV is very similar to the LOV domains in NPH1, a plasma membrane-associated blue light receptor kinase that regulates phototropism (Huala, E., Oeller, P.W., Liscum, E., Han, I-S., Larsen, E. & Briggs, W.R. (1997) Science, 278, 2120-2123). LKP1 mRNA accumulates in roots, stems, flowers and siliques. It is most abundant in leaves, and least abundant in seeds. Transgenic plants with a beta-glucuronidase (GUS) reporter gene driven by a 1.5 kb LKP1 promoter display strong GUS activity in leaves. Transgenic plants with a 35S:LKP1 cDNA gene overexpress LKP1 mRNA. These plants have elongated hypocotyls and petioles with elongated cells, and exhibit distinct cotyledon movement during the day. Expression of 35S:LKP1 in transgenic Arabidopsis promotes late flowering in plants grown under long-day, but not under short-day conditions. Vernalization does not affect the late flowering phenotype of the 35S:LKP1 plants. Transgenic plants possessing the 35S:GFP-LKP1 construct also have long hypocotyles and petioles, and a late flowering phenotype, suggesting that the GFP-LKP1 fusion protein is active. The GFP-associated fluorescence in 35S:GFP-LKP1 plants is observed in nuclei and cytosol, indicating that LKP1 is a new nucleo-cytoplasmic factor that influences flowering time in the long day pathway of Arabidopsis.     

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23^fyp and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.     

Causes of Flowering of Long-Day Potato Species under Short-Day and Cold-Night Conditions

Flowering and tuber formation in high-mountain potato species Solanum sparsipilum Bitt., S. acaule Bitt., S. punae Juz., S. demissum Lindl., and a tuber crop Ullucus tuberosus Caldas. were investigated. All these species are characterized by absolute requirement of long day-length for flowering and short day-length for tuberization. Plants were grown under the following conditions: natural day-length with a photoperiod of 17 h or longer (treatment 1), short days with a photoperiod of 12 h and warm nights (15–20°C) (treatment 2), and short days with cold nights (5–6°C) (treatment 3). In the first treatment, plants produced flowers but no tubers. In the second treatment, plants produced tubers but no flowers. In the third treatment, plants produced both flowers and tubers. In leaves of S. acaule and U. tuberosus, the levels of gibberellins and ABA were determined. A high activity of gibberellins in the third treatment was similar to that in the first treatment, whereas high ABA activity in the third treatment was similar to that in the second treatment. It is supposed that cold nights retard the destruction of GA in plants during the dark period of diurnal cycle and ensure a permanently high level of gibberellins, which facilitates flowering of long-day species under short-day conditions. The high level of ABA is considered a plant response to short-day conditions, which is favorable for tuberization.     

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the <Emphasis Type="Italic">PHYB</Emphasis> Gene from <Emphasis Type="Italic">Arabidopsis</Emphasis>

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.     

LOV KELCH PROTEIN2 and ZEITLUPE repress Arabidopsis photoperiodic flowering under non-inductive conditions, dependent on FLAVIN-BINDING KELCH REPEAT F-BOX1

LOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN‐BINDING KELCH REPEAT F‐BOX1 (FKF1) constitute a family of Arabidopsis F‐box proteins that regulate the circadian clock. Over‐expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over‐expressing LKP2, CO and FT expression was down‐regulated under long‐day conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Over‐expression of LKP2 Kelch repeats induced late flowering under long‐day conditions. lkp2 ztl double mutant plants flowered earlier than wild‐type plants under short‐day (non‐inductive) conditions, and both CO and FT expression levels were up‐regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non‐inductive conditions, and this is dependent on FKF1.     

Expression of AtECA3 in tobacco modifies its responses to manganese, zinc and calcium

Mn- and Zn-deficiency or excess reduce plant growth and development. Engineering plants with enhanced metal tolerance and accumulation is a major goal in phytoremediation/phytostabilization. Moreover, improved growth under unfavourable mineral conditions contributes to better crop production. In this study, ECA3 cDNA from Arabidopsis thaliana, encoding a P_2A-ATPase, was introduced into Nicotiana tabacum var. Xanthi, to examine its value for modifying responses to cations, primarily Mn, Zn and Ca. AtECA3 was ectopically expressed under the CaMV 35S promoter. Transgenic and wild-type plants were tested under hydroponic conditions for their responses to a range of metal exposures (low, moderate and high concentrations), and their tolerance and accumulation were evaluated. AtECA3 expression resulted in better growth of plants at moderate levels of Mn (2 μM) in the medium and enhanced tolerance to high Mn (100 μM). Transgenic plants were also more tolerant to Ca-deficiency conditions, although they showed no differences to wild-type with respect to overall Ca levels. Transgene expression did not produce one unique pattern of Mn and Zn accumulation but instead depended on the external concentration of the particular metal supplied. Thus the enhancement of plant productivity at moderate Mn levels and increased Mn tolerance at high (toxic) Mn supply, as well as the slight increase in Ca-deficiency tolerance seen in ECA3-transformed plants indicates that this gene could be useful in plant biotechnological strategies.     

Tuber Formation and Growth of in vitro Cultivated Transgenic Potato Plants Overproducing Phytochrome B

We studied the effect of the ectopic expression of the Arabidopsis PHYB gene, which encodes the phytochrome B (phyB) apoprotein, under the control of cauliflower mosaic virus 35S promoter on the photoperiodic response of tuberization and growth of potato (Solanum tuberosum L., cv. Désirée) transformed lines. Stem cuttings of transformed and control plants were cultured on Murashige and Skoog nutrient medium containing 5 or 8% sucrose in the phytotron chambers at 20°C under conditions of a long day (16 h), a short day (10 h), or in darkness. We showed that the overexpression of the PHYB gene enhanced the inhibitory effect of the long day on tuberization. In addition, tuber initiation in these transformed plants occurred at a higher sucrose concentration. The insertion of the PHYB gene decreased plant and tuber weights and shortened stems and internodes. Thus, we demonstrated the complex result of the PHYB gene insertion: it affected the photoperiodic response of tuberization, the control of tuber initiation by sucrose, and the growth of potato vegetative organs.     

Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics

GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA₁ and GA₄. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato.     

Photoperiodic response of larvae of the yellow-spotted longicorn beetle Psacothea hilaris after removal of the stemmata

The role of the stemmata in photoperiodism has been examined in holometabolic insects, but the only reliable results in Coleoptera have been obtained in Leptocarabus kumagaii (Carabidae), the larvae of which do not respond to photoperiod without stemmata. In the present study, photoperiodism was examined in another coleopteran, Psacothea hilaris (Pascoe) (Cerambycidae), after surgical removal of the stemmata. Larvae reared under short-day conditions and transferred to long-day conditions on day 2 of the 5th instar pupated without further larval molts, whereas those continuously reared under short-day conditions underwent supernumerary molts and did not pupate. When the stemmata were removed on day 2 of the 5th instar, the larvae pupated under long-day conditions but did not do so under short-day conditions. However, under long-day conditions some underwent supernumerary molts before pupation. Larvae from which the sensilla trichodeum were removed showed a similar response to that of stemmata-deficient larvae, and larvae from which stemmata were removed at a younger stage (day 2 of the 4th instar) responded to photoperiod similarly to intact larvae. Thus, supernumerary molts under long-day conditions after removal of the stemmata were attributed to injury due to surgery, rather than a change in photoperiodic photoreception. Therefore, we conclude that larvae of P. hilaris show a photoperiodic response after removal of stemmata, in contrast to larvae of L. kumagaii.     

干旱胁迫对高山柳和沙棘幼苗光合生理特征的影响

为了解干旱河谷-山地森林交错带植物光合生理特征对干旱胁迫的响应。以交错带两种典型植物高山柳(Salix paraqplesia)和沙棘(Hippophae rhamnoides)为研究对象,研究其在不同程度的干旱胁迫条件下植株气体交换参数的日变化特征。干旱胁迫显著降低了两种植物叶片数、叶面积、叶片生物量、比叶面积、色素含量、净光合速率(Pn)、气孔导度(gs)和气孔限制值(Ls)等与光合生理过程密切相关的叶片指标,但增大了胞间CO₂浓度(Ci)和内禀水分利用效率(WUEi)。植物叶片的气体交换参数(如:Pn和gs)日变化并未完全随着光合有效辐射的增强和温度的升高而增加,全天以11:00最大,"午休"现象明显。相对而言,沙棘在干旱胁迫条件下表现出相对较高的叶面积、Pn、gs和WUEi,具有相对更强的适应干旱环境的能力,而高山柳对干旱胁迫更为敏感。     

干旱胁迫对高山柳和沙棘幼苗光合生理特征的影响

为了解干旱河谷-山地森林交错带植物光合生理特征对干旱胁迫的响应。以交错带两种典型植物高山柳(Salix paraqplesia)和沙棘(Hippophae rhamnoides)为研究对象,研究其在不同程度的干旱胁迫条件下植株气体交换参数的日变化特征。干旱胁迫显著降低了两种植物叶片数、叶面积、叶片生物量、比叶面积、色素含量、净光合速率(Pn)、气孔导度(gs)和气孔限制值(Ls)等与光合生理过程密切相关的叶片指标,但增大了胞间CO₂浓度(Ci)和内禀水分利用效率(WUEi)。植物叶片的气体交换参数(如:Pn和gs)日变化并未完全随着光合有效辐射的增强和温度的升高而增加,全天以11:00最大,"午休"现象明显。相对而言,沙棘在干旱胁迫条件下表现出相对较高的叶面积、Pn、gs和WUEi,具有相对更强的适应干旱环境的能力,而高山柳对干旱胁迫更为敏感。