BrPARP1, a Poly (ADP-Ribose) Polymerase Gene, Is Involved in Root Development in Brassica rapa under Drought Stress

PARP proteins are highly conserved homologs among the eukaryotic poly (ADP-ribose) polymerases. After activation, ADP-ribose polymers are synthesized on a series of ribozymes that use NAD+ as a substrate. PARPs participate in the regulation of various important biological processes, such as plant growth, development, and stress response. In this study, we characterized the homologue of PARP1 in B. rapa using RNA interference (RNAi) to reveal the underlying mechanism responding to drought stress. Bioinformatics and expression pattern analyses demonstrated that two copy numbers of PARP1 genes (BrPARP1.A03 and BrPARP1.A05) in B. rapa following a whole-genome triplication (WGT) event were retained compared with Arabidopsis, but only BrPARP1.A03 was predominantly transcribed in plant roots. Silencing of BrPARP1 could markedly promote root growth and development, probably via regulating cell division, and the transgenic Brassica lines showed more tolerance under drought treatment, accompanied with substantial alterations including accumulated proline contents, significantly reduced malondialdehyde, and increased antioxidative enzyme activity. In addition, the findings showed that the expression of stress-responsive genes, as well as reactive oxygen species (ROS)-scavenging related genes, was largely reinforced in the transgenic lines under drought stress. In general, these results indicated that BrPARP1 likely responds to drought stress by regulating root growth and the expression of stress-related genes to cope with adverse conditions in B. rapa.

Overexpression of miR5505 enhanced drought and salt resistance in rice (Orayza sativa)

Rice is one of the most important crops in the world and half of the world population consumes it as their staple food. The abiotic stresses caused by drought, salt and other stresses have severely impacted rice production. MicroRNAs (miRNAs) are a type of small non-coding RNAs which widely reported as gene regulators, suppressing genes expression by degradation mRNA or translation inhibition. Previously, high-throughput sequencing has found a conserved miRNA miR5505 responding to drought stress in Dongxiang wild rice (DXWR). Several other studies also revealed that miR5505 was involved in rice stress responses. We further studied the effect of miRNA in drought and salt tolerance by overexpression it in rice. 2 in 18 successfully transformed transgenic lines with higher miR5505 expression were selected and then drought and salt resistance ability were evaluated. Both transgenic lines showed stronger drought and salt tolerance than wild-type (WT). Putative targets of miR5505 were identified by psRNATarget and several of them were found stress-related. RNA-seq found 1,980 differentially expressed genes (DEGs) in transgenic lines. Among them, 978 genes were down-regulated. Three genes were predicted by psRNATarget and two of them might be stress-related. We also found various environmental stress cis-acting elements in upstream of miR5505 promoter through Software PlantCARE. In all, we improved rice drought and salt tolerance by overexpressing miR5505, and the generated putative targets and cis-acting elements also suggested miR5505 might play important roles in the regulation of drought and salt responses. Keywords: rice, overexpression line , drought and salt stress, miR5505

Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a

Overexpression of Arabidopsis Dehydration Response Element Binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. While the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be colocalized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding when compared to non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a overexpressing lines, suggesting antagonism between heat and dehydration stress pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and rapid response towards stress tolerance as well as attenuation of the signal to prevent deleterious effects of the runoff response.

Isolation and Characterization of Extracellular Vesicles from the Fungal Phytopathogen Colletotrichum higginsianum

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic lines expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.

Cryopreservation of Anopheles stephensi embryos

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

Stability of Transgene Inheritance in Progeny of Field-Grown Pear Trees over a 7-Year Period

Breeding woody plants is a very time-consuming process, and genetic engineering tools have been used to shorten the juvenile phase. In addition, transgenic trees for commercial cultivation can also be used in classical breeding, but the segregation of transgenes in the progeny of perennial plants, as well as the possible appearance of unintended changes, have been poorly investigated. We studied the inheritance of the uidA gene in the progeny of field-grown transgenic pear trees for 7 years and the physical and physiological parameters of transgenic seeds. A total of 13 transgenic lines were analyzed, and the uidA gene segregated 1:1 in the progeny of 9 lines and 3:1 in the progeny of 4 lines, which is consistent with Mendelian inheritance for one and two transgene loci, respectively. Rare and random deviations from the Mendelian ratio were observed only for lines with one locus. Transgenic seeds’ mass, size, and shape varied slightly, despite significant fluctuations in weather conditions during cultivation. Expression of the uidA gene in the progeny was stable. Our study showed that the transgene inheritance in the progeny of pear trees under field conditions occurs according to Mendelian ratio, does not depend on the environment, and the seed vigor of transgenic seeds does not change.

Different Fruit-Specific Promoters Drive AtMYB12 Expression to Improve Phenylpropanoid Accumulation in Tomato

Tomato is an economically crucial vegetable/fruit crop globally. Tomato is rich in nutrition and plays an essential role in a healthy human diet. Phenylpropanoid, a critical compound in tomatoes, reduces common degenerative and chronic diseases risk caused by oxidative stress. As an MYB transcription factor, ATMYB12 can increase phenylpropanoid content by activating phenylpropanoid synthesis related genes, such as PAL, C4H, 4CL, CHS. However, the heterologous expression of AtMYB12 in tomatoes can be altered through transgenic technologies, such as unstable expression vectors and promoters with different efficiency. In the current study, the efficiency of other fruit-specific promoters, namely E8S, 2A12, E4, and PG, were compared and screened, and we determined that the expression efficiency of AtMYB12 was driven by the E8S promoter was the highest. As a result, the expression of phenylpropanoid synthesis related genes was regulated by AtMYB12, and the phenylpropanoid accumulation in transgenic tomato fruits increased 16 times. Additionally, the total antioxidant capacity of fruits was measured through Trolox equivalent antioxidant capacity (TEAC) assay, which was increased by 2.4 times in E8S transgenic lines. TEAC was positively correlated with phenylpropanoid content. Since phenylpropanoid plays a crucial role in the human diet, expressing AtMYB12 with stable and effective fruit-specific promoter E8S could improve tomato’s phenylpropanoid and nutrition content and quality. Our results can provide genetic resources for the subsequent improvement of tomato varieties and quality, which is significant for human health.

Asymmetric wall ingrowth deposition in Arabidopsis phloem parenchyma transfer cells is tightly associated with sieve elements

In Arabidopsis, polarized deposition of wall ingrowths in phloem parenchyma (PP) transfer cells (TCs) occurs adjacent to cells of the sieve element/companion cell (SE/CC) complex. However, the spatial relationships between these different cell types in minor veins, where phloem loading occurs, are poorly understood. PP TC development and wall ingrowth localization were compared to other phloem cells in leaves of Col-0 and the transgenic lines AtSUC2::AtSTP9-GFP and AtSWEET11::AtSWEET11-GFP that identify CCs and PP respectively. The development of PP TCs in minor veins, indicated by deposition of wall ingrowths, proceeded basipetally in leaves. However, not all PP develop ingrowths and higher levels of wall ingrowth deposition occur in abaxial- compared to adaxial-positioned PP TCs. Furthermore, the deposition of wall ingrowths was exclusively initiated on and preferentially covered the PP TC/SE interface, rather than the PP TC/CC interface, and only occurred in PP cells that were adjacent to SEs. Collectively, these results demonstrate the dominant impact of SEs on wall ingrowth deposition in PP TCs and suggest the existence of two sub-types of PP cells in leaf minor veins. Compared to PP cells, PP TCs showed more abundant accumulation of AtSWEET11-GFP, indicating functional differences in phloem loading between PP and PP TCs.

Dephosphorylation of Nitrate Reductase Protein Regulates Growth of Rice and Adaptability to Low Temperature

Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and its activity is regulated by post-translational phosphorylation. To investigate the effect of NIA1 protein dephosphorylation on the growth of rice and its adaptability to low temperature, we analyzed phenotype, chlorophyll content, nitrogen utilization, and antioxidant capacity at low temperature in lines with a mutated NIA1 phosphorylation site (S532D and S532A), an OsNia1 over-expression line (OE), and wild-type Kitaake rice (WT). Plant height, dry matter weight, and chlorophyll content of S532D and S532A were lower than those of WT and OE under normal growth conditions but were higher than those of WT and OE at low temperature. Compared with WT and OE, the nitrite, H2O2, and MDA contents of S532D and S532A leaves were higher under normal growth conditions. The difference in leaf nitrite content between transgenic lines and WT was narrower at low temperature, especially in S532D and S532A, while H2O2 and MDA contents of S532D and S532A leaves were lower than those in WT and OE leaves. The NH4+-N and amino acid contents of S532D and S532A leaves were higher than those of WT and OE leaves under normal or low temperature. qRT-PCR results revealed that transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were positively correlated with those of OsNia1, and the transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were significantly higher in transgenic lines than in WT under both normal and low temperature. Phosphorylation of NR is a steady-state regulatory mechanism of nitrogen metabolism, and dephosphorylation of NIA1 protein improved NR activity and nitrogen utilization efficiency in rice. Excessive accumulation of nitrite under normal growth conditions inhibits the growth of rice; however, accumulation of nitrite is reduced at low temperature, enhancing the cold tolerance of rice.

Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.