<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.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Overexpression of wheat <Emphasis Type="Italic">TaNCED</Emphasis> gene in <Emphasis Type="Italic">Arabidopsis</Emphasis> enhances tolerance to drought stress and delays seed germination

Abscisic acid (ABA) regulates various plant physiological processes, especially participates in the plant responses to harsh environments. The 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis pathway. Here, a TaNCED with an 1 887-bp open reading frame was cloned from wheat, which encodes a peptide of 628 amino acids. A chloroplast transit peptide sequence was found at the N-terminus of the TaNCED protein. Multiple sequence alignments indicate that the TaNCED protein shared high similarities with other NCEDs from different species. Real-time quantitative PCR analysis shows that expression of TaNCED was strongly up-regulated by treatments with ABA, polyethylene glycol, and drought stress, and it was down-regulated during germination of the wheat seeds. Ectopic overexpression of the TaNCED gene in Arabidopsis resulted in an increase of endogenous ABA and free proline content. A lower water loss rate and stomatal conductance of leaves were found in the transgenic plants in comparison with the wild type. Subsequently, the transgenic plants displayed an enhanced tolerance to drought stress but delayed seed germination. These data provide evidence that the TaNCED might play a primary role in regulation of ABA content during water stress and seed dormancy.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

Effects of <Emphasis Type="Italic">Prangos ferulacea</Emphasis> aqueous and hydroalcoholic extracts obtained from different organs on the regeneration of <Emphasis Type="Italic">Trifolium resupinatum</Emphasis>

Prangos ferulacea is one of the widely used, nutritional and popular fodders in livestock industry. This species is also considered as an important option in rangeland restoration and management. In this study, the comparative phytotoxic activity of aqueous and hydroalcoholic extracts obtained from different organs (flower, shoot and leaf) of P. ferulacea on proline content, seed germination and seedling growth of Trifolium resupinatum has been investigated. According to the results, the hydroalcoholic extract of P. ferulaceae flower possesses the highest total phenolic and flavonoid content and the uppermost phytotoxic effect on T. resupinatum. The extracts significantly decreased seed germination and seedling growth of T. resupinatum and increased the proline content. Our findings indicate that hydroalcoholic extract induced a stronger oxidative stress in T. resupinatum. Finally, based on the results, aqueous allelochemicals that originated from P. ferulacea played a significant role in the successful propagation and development of T. resupinatum in rehabilitated pastures. According to our results, the phytotoxicity effect of the hydroalcoholic extract was significantly higher than that of the aqueous extract. Since in nature, the allelopathic interaction between plants is closer to the aqueous method, primary evaluations of rangeland restoration using this method is suggested.