Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.     

Photoperiodic flower induction in <Emphasis Type="Italic">Ipomoea nil</Emphasis> is accompanied by decreasing content of gibberellins

The involvement of gibberellins (GAs) in the control of flower induction in the short-day plant Ipomoea nil has been investigated. To clarify the molecular basis of this process, we identified the full-length cDNAs of the InGA20ox3 and InGA2ox1 genes, which encode enzymes responsible for GA biosynthesis and catabolism, respectively. We studied the expression patterns of both genes and determined the tissue and cellular immunolocalisation of gibberellic acid (GA₃) in the cotyledons of 5-day-old seedlings growing under inductive and non-inductive photoperiodic conditions. In the second half of the inductive night, which is crucial for flower induction in I. nil, InGA20ox3 expression decreased, whereas InGA2ox1 mRNA accumulated, which indicates that photoperiod regulates the activity of both genes. Furthermore, these changes are correlated with GA₃ level. Thus, our results support the thesis that the proper balance between the expression of the InGA20ox3 and InGA2ox1 genes and low GA₃ content correlate with photoperiodic flower induction in I. nil.     

<Emphasis Type="Italic">CKB1</Emphasis> is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.     

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.     

Rice inositol polyphosphate kinase gene (<Emphasis Type="Italic">OsIPK2</Emphasis>), a putative new player of gibberellic acid signaling,involves in modulation of shoot elongation and fertility

Gibberellic acid (GA) is an important plant hormone mediating plant growth and development throughout the life span. Although many GA biosynthesis genes and signaling components have been revealed, the signal transduction mechanisms from GA perception to physiological actions are still largely unclear. In this study, we investigated the functions of a rice (Oryza sativa) inositol polyphosphate kinase gene (OsIPK2) in rice growth and development, showing that OsIPK2 is a putative new player in GA signaling. OsIPK2 is widely expressed in rice with high accumulation in tender and rapidly dividing tissues. The OsIPK2 protein is mainly localized in the nucleus and plasma membrane. To study the biological roles of OsIPK2 in rice, RNA interference and overexpression transgenic plants were generated. OsIPK2 antisense plants exhibited taller seedling height and lower fertility rate than the wild type, while overexpression lines showed reduced plant height. Microarray and qRT-PCR assays showed that expression levels of several GA-related genes were altered in transgenic plants. Besides, down-regulation of OsIPK2 resulted in hypersensitivity to paclobutrazol (PAC), a GA biosynthesis inhibitor. We also described that the expression of OsIPK2 could be either induced by GA or repressed by PAC. Taken together, these findings suggested that OsIPK2 is likely a negative regulator of GA signaling and involves in modulating shoot elongation and fertility.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

Characterization of <Emphasis Type="Italic">PP2A</Emphasis>-<Emphasis Type="Italic">A3</Emphasis> mRNA expression and growth patterns in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under drought stress and abscisic acid

Phosphoprotein phosphatase 2A (PP2A) plays a crucial role in cellular processes via reversible dephosphorylation of proteins. The activity of this enzyme depends on its subunits. There is little information about mRNA expression of each subunit and the relationship between these gene expressions and the growth patterns under stress conditions and hormones. Here, mRNA expression of subunit A3 of PP2A and its relationship with growth patterns under different levels of drought stress and abscisic acid (ABA) concentration were analyzed in Arabidopsis thaliana. The mRNA expression profiles showed different levels of the up- and down-regulation of PP2AA3 in roots and shoots of A. thaliana under drought conditions and ABA treatments. The results demonstrated that the regulation of PP2AA3 expression under the mentioned conditions could indirectly modulate growth patterns such that seedlings grown under severe drought stress and those grown under 4 µM ABA had the maximum number of lateral roots and the shortest primary roots. In contrast, the minimum number of lateral roots and the longest primary roots were observed under mild drought stress and 0.5 µM ABA. Differences in PP2AA3 mRNA expression showed that mechanisms involved in the regulation of this gene under drought conditions would probably be different from those that regulate the PP2AA3 expression under ABA. Co-expression of PP2AA3 with each of PIN1-4,7 (PP2A activity targets) depends on the organ type and different levels of drought stress and ABA concentration. Furthermore, fluctuations in the PP2AA3 expression proved that this gene cannot be suitable as a reference gene although PP2AA3 is widely used as a reference gene.     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.