A mutation in Arabidopsis SAL1 alters its in vitro activity against IP3 and delays developmental leaf senescence in association with lower ROS levels.

KEY MESSAGE: Our manuscript is the first to find a link between activity of SAL1/OLD101 against IP3 and plant leaf senescence regulation and ROS levels assigning a potential biological role for IP3. Leaf senescence is a genetically programmed process that limits the longevity of a leaf. We identified and analyzed the recessive Arabidopsis stay-green mutation onset of leaf death 101 (old101). Developmental leaf longevity is extended in old101 plants, which coincided with higher peroxidase activity and decreased H2O2 levels in young 10-day-old, but not 25-day-old plants. The old101 phenotype is caused by a point mutation in SAL1, which encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity. SAL1 activity is highly specific for its substrates 3-polyadenosine 5-phosphate (PAP) and inositol 1, 4, 5-trisphosphate (IP3), where it removes the 1-phosphate group from the IP3 second messenger. The in vitro activity of recombinant old101 protein against its substrate IP3 was 2.5-fold lower than that of wild type SAL1 protein. However, the in vitro activity of recombinant old101 mutant protein against PAP remained the same as that of the wild type SAL1 protein. The results open the possibility that the activity of SAL1 against IP3 may affect the redox balance of young seedlings and that this delays the onset of leaf senescence.

A novel, simple, and stable mesoporous silica nanoparticle-based gene transformation approach in Solanum lycopersicum.

In this study, a novel and stable gene transformation system was developed under control of Maize Proteinase Inhibitor (MPI) as an inducible promoter using the Mesoporous Silica Nanoparticles (MSNs). The functionalized MSNs with a proper particle size were synthesized and attached to a recombinant construct (pDNA) containing cryIAb gene under the control of MPI promoter (pPZP122:MPI:cryIAb:MSN [pDNA: MSN]) following transformation of tomato plants through injection of the pDNA: MSN complex into tomato red fruit at early ripening stage and then, putative transgenic seeds were collected. As an initial selection, gentamicin-resistant seedlings of T1 (24.24%) and T2 (61.37%) plants were identified. The transgene integration and expression were confirmed through the PCR, RT-PCR, and western blot approaches in the selected seedlings. PCR analysis showed that transformation frequency was equal to 10.71% in T1 plants. Semi-quantitative RT-PCR analysis confirmed the transcript expression of cryIAb in all the T1 and T2 PCR-positive plants. Western blot analysis confirmed the existence of CryIAb protein in the leaves of T2 putative transgenic plants. Accordingly, the results demonstrated that the transgene has more likely integrated into the tomato genome through homologous recombination. Bioassay was carried out for further assessment of the plant responses to Tuta absoluta resulting in an enhanced tolerance of the plant. In conclusion, the MSN-mediated stable transformation system under the MPI as an inducible promoter can be used as a suitable alternative for conventional genetic transformation methods due to its biodegradability, biocompatibility, cost and time-effectiveness, and positive effect on the plant defense against pathogens and pests.

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco.

Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin-binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD-DrsB1 and pGSA/DrsB1-CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi-quantitative RT-PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD-DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1-CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

Correction to: Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity.

The original version of this article unfortunately contained a mistake in the unit "µg/l". The unit "µg/l" should be corrected to "µg/ml" throughout the paper.

Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity.

Expression of strong antimicrobial peptides in plants is of great interest to combat a wide range of plant pathogens. To bring the Dermaseptin B1 (DrsB1) peptide to the intimate contact of the plant pathogens cell wall surface, the DrsB1 encoding sequence was fused to the C-terminal part of the two copies of the chitin-binding domain (CBD) of the Avr4 effector protein and used for Agrobacterium rhizogenes-mediated transformation. The expression of the recombinant protein in the tobacco hairy roots (HRs) was confirmed by molecular analysis. Antimicrobial activity analysis of the recombinant protein purified from the transgenic HRs showed that the (CBD)2-DrsB1 recombinant protein had a significant (p < 0.01) antimicrobial effect on the growth of different fungal and bacterial pathogens. The results of this study indicated that the recombinant protein had a higher antifungal activity against chitin-producing Alternaria alternata than Pythium spp. Scanning electron microscopy images demonstrated that the recombinant protein led to fungal hypha deformation, fragmentation, and agglutination of growing hypha, possibly by dissociating fungal cell wall components. In vitro evidences suggest that the expression of the (CBD)2-DrsB1 recombinant protein in plants by generating transgenic lines is a promising approach to produce disease-resistant plants, resistance to chitin-producing pathogenic fungi.

Systematic analysis of NAC transcription factors' gene family and identification of post-flowering drought stress responsive members in sorghum.

KEY MESSAGE: SbNAC genes (131) encoding 183 proteins were identified from the sorghum genome and characterized. The expression patterns of SbSNACs were evaluated at three sampling time points under post-flowering drought stress. NAC proteins are specific transcription factors in plants, playing vital roles in development and response to various environmental stresses. Despite the fact that Sorghum bicolor is well-known for its drought-tolerance, it suffers from grain yield loss due to pre and post-flowering drought stress. In the present study, 131 SbNAC genes encoding 183 proteins were identified from the sorghum genome. The phylogenetic trees were constructed based on the NAC domains of sorghum, and also based on sorghum with Arabidopsis and 8 known NAC domains of other plants, which classified the family into 15 and 19 subfamilies, respectively. Based on the obtained results, 13 SbNAC proteins joined the SNAC subfamily, and these proteins are expected to be involved in response to abiotic stresses. Promoter analysis revealed that all SbNAC genes comprise different stress-associated cis-elements in their promoters. UTRs analysis indicated that 101 SbNAC transcripts had upstream open reading frames, while 39 of the transcripts had internal ribosome entry sites in their 5'UTR. Moreover, 298 miRNA target sites were predicted to exist in the UTRs of SbNAC transcripts. The expression patterns of SbSNACs were evaluated in three genotypes at three sampling time points under post-flowering drought stress. Based on the results, it could be suggested that some gene members are involved in response to drought stress at the post-flowering stage since they act as positive or negative transcriptional regulators. Following further functional analyses, some of these genes might be perceived to be promising candidates for breeding programs to enhance drought tolerance in crops.

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in Arabidopsis.

BACKGROUND: Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified. RESULTS: The seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1) mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0) and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant) ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession) and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress. CONCLUSIONS: The cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell death regulation.