Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Pepper U-Box E3 ubiquitin ligase 24, CaPUB24, negatively regulates drought stress response.

Under stress conditions, plants modulate their internal states and initiate various defence mechanisms to survive. The ubiquitin-proteasome system is one of the critical modules in these mechanisms, and Plant U-Box proteins play an important role in this process as E3 ubiquitin ligases. Here, we isolated the Plant U-box 24 gene CaPUB24 (Capsicum annuum Plant U-Box 24) from pepper and characterized its functions in response to drought stress. We found that, compared to the other CaPUBs in the same group, the expression of CaPUB24 was significantly induced by drought stress. We also found that CaPUB24 was localized to the nucleus and cytoplasm and had E3 ubiquitin ligase activity. To investigate the biological role of CaPUB24 in response to drought stress further, we generated CaPUB24-silenced pepper plants and CaPUB24-overexpressing Arabidopsis transgenic plants. CaPUB24-silenced pepper plants exhibited enhanced drought tolerance compared to the control plants due to reduced transpirational water loss and increased abscisic acid (ABA) sensitivity. In contrast, CaPUB24-overexpressing Arabidopsis transgenic plants exhibited reduced drought tolerance and ABA-insensitive phenotypes. Our findings suggest that CaPUB24 negatively modulates drought stress response in an ABA-dependent manner.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Understanding the role of the fructose-1,6-bisphosphatase gene for enhancing the photosynthetic rate in Arabidopsis thaliana.

Transgenic Arabidopsis thaliana (ecotype Columbia) was successfully transformed with the gene fructose-1,6-bisphosphatase (FBPas e) and named as AtFBPase plants. Transgenic plants exhibited stable transformation, integration and significantly higher expressions for the transformed gene. Morphological evaluation of transgenic plants showed increased plant height (35cm), number of leaves (25), chlorophyll contents (28%), water use efficiency (increased from 1.5 to 2.6µmol CO2 µmol-1 H2 O) and stomatal conductance (20%), which all resulted in an enhanced photosynthetic rate (2.7µmolm-2 s-1 ) compared to wild type plants. This study suggests the vital role of FBPase gene in the modification of regulatory pathways to enhance the photosynthetic rate, which can also be utilised for economic crops in future.

Overexpression of soybean GmDHN9 gene enhances drought resistance of transgenic Arabidopsis.

Soybean is one of the important oil crops and a major source of protein and lipids. Drought can cause severe soybean yields. Dehydrin protein (DHN) is a subfamily of LEA proteins that play an important role in plant responses to abiotic stresses. In this study, the soybean GmDHN9 gene was cloned and induced under a variety of abiotic stresses. Results showed that the GmDHN9 gene response was more pronounced under drought induction. Subcellular localization results indicated that the protein was localized in the cytoplasm. The role of transgenic Arabidopsis plants in drought stress response was further studied. Under drought stress, the germination rate, root length, chlorophyll, proline, relative water content, and antioxidant enzyme content of transgenic Arabidopsis thaliana transgenic genes were higher than those of wild-type plants, and transgenic plants contained less O2-, H2O2 and MDA contents. In short, the GmDHN9 gene can regulate the homeostasis of ROS and enhance the drought resistance of plants.

AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

Maize ZmLAZ1-3 gene negatively regulates drought tolerance in transgenic Arabidopsis.

BACKGROUND: Molecular mechanisms in response to drought stress are important for the genetic improvement of maize. In our previous study, nine ZmLAZ1 members were identified in the maize genome, but the function of ZmLAZ1 was largely unknown. RESULTS: The ZmLAZ1-3 gene was cloned from B73, and its drought-tolerant function was elucidated by expression analysis in transgenic Arabidopsis. The expression of ZmLAZ1-3 was upregulated by drought stress in different maize inbred lines. The driving activity of the ZmLAZ1-3 promoter was induced by drought stress and related to the abiotic stress-responsive elements such as MYB, MBS, and MYC. The results of subcellular localization indicated that the ZmLAZ1-3 protein localized on the plasma membrane and chloroplast. The ectopic expression of the ZmLAZ1-3 gene in Arabidopsis significantly reduced germination ratio and root length, decreased biomass, and relative water content, but increased relative electrical conductivity and malondialdehyde content under drought stress. Moreover, transcriptomics analysis showed that the differentially expressed genes between the transgenic lines and wild-type were mainly associated with response to abiotic stress and biotic stimulus, and related to pathways of hormone signal transduction, phenylpropanoid biosynthesis, mitogen-activated protein kinase signaling, and plant-pathogen interaction. CONCLUSION: The study suggests that the ZmLAZ1-3 gene is a negative regulator in regulating drought tolerance and can be used to improve maize drought tolerance via its silencing or knockout.

Spatiotemporal formation of glands in plants is modulated by MYB-like transcription factors.

About one third of vascular plants develop glandular trichomes, which produce defensive compounds that repel herbivores and act as a natural biofactory for important pharmaceuticals such as artemisinin and cannabinoids. However, only a few regulators of glandular structures have been characterized so far. Here we have identified two closely-related MYB-like genes that redundantly inhibit the formation of glandular cells in tomatoes, and they are named as GLAND CELL REPRESSOR (GCR) 1 and 2. The GCR genes highly express in the apical cells of tomato trichomes, with expression gradually diminishing as the cells transition into glands. The spatiotemporal expression of GCR genes is coordinated by a two-step inhibition process mediated by SlTOE1B and GCRs. Furthermore, we demonstrate that the GCR genes act by suppressing Leafless (LFS), a gene that promotes gland formation. Intriguingly, homologous GCR genes from tobacco and petunia also inhibit gland formation, suggesting that the GCR-mediated repression mechanism likely represents a conserved regulatory pathway for glands across different plant species.

Resistance management and integrated pest management insights from deployment of a Cry3Bb1+ Gpp34Ab1/Tpp35Ab1 pyramid in a resistant western corn rootworm landscape.

In Nebraska USA, many populations of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, now exhibit some level of resistance to all corn rootworm-active Bacillus thuringiensis Berliner (Bt) proteins expressed in commercial hybrids. Therefore, a study was conducted in northeast Nebraska from 2020-2022 to reevaluate current corn rootworm management options in continuous maize (consecutive planting for ≥2 years). Results from on-farm experiments to evaluate a standard soil-applied insecticide (Aztec® 4.67G) in combination with non-rootworm Bt or rootworm-active Bt pyramided maize (Cry3Bb1 + Gpp34Ab1/Tpp35Ab1) are reported within the context of WCR Bt resistance levels present. Corrected survival from Bt pyramid single-plant bioassays (<0.3, 0.3-0.49, >0.5) was used to place populations into 3 resistance categories. Variables evaluated included root injury, adult emergence, proportion lodged maize, and grain yield. Key results: A composite analysis of all populations across resistance levels indicated that addition of soil insecticide to Bt pyramid significantly reduced adult emergence and lodging but did not significantly increase root protection or yield. Within and among resistance category analyses of root injury revealed that the Bt pyramid remained highly efficacious at any non-rootworm Bt root injury level when resistance was absent or low. When corrected survival was >0.3, mean Bt pyramid root injury tracked more closely in a positive linear fashion with mean non-rootworm Bt root injury (rootworm density x level of resistance interaction). Similar trends were obtained for adult emergence but not yield. Mean Bt pyramid root injury rating was <0.75 in most populations with Bt resistance, which contributed to no significant yield differences among categories. Results are discussed within the context of IPM:IRM tradeoffs and the need to reduce WCR densities in this system to decrease the impact of the density x resistance interaction to bridge use of current pyramids with new technologies introduced over the next decade.

Heterologous expression of taxane genes confers resistance to fall armyworm in Nicotiana benthamiana.

KEY MESSAGE: Taxadiene synthase, taxadiene-5α-hydroxylase, and taxane 13α-hydroxylase genes were introduced into Nicotiana benthamiana, and the improved resistance to lepidoptera pest fall armyworm was reported. Fall armyworm (FAW) is a serious agricultural pest. Genetic engineering techniques have been used to create pest-resistant plant varieties for reducing pest damage. Paclitaxel is a diterpenoid natural metabolite with antineoplastic effects in medicine. However, the effects of taxanes on the growth and development of lepidoptera pests, such as the FAW, are unknown. Here, selected paclitaxel precursor biosynthesis pathway genes, taxadiene synthase, taxane 5α-hydroxylase, and taxane 13α-hydroxylase, were engineered in the heterologous host Nicotiana benthamiana plants. Bioassay experiments showed that the transgenic N. benthamiana plants displayed improved resistance to FAW infestation, with degeneration of gut tissues and induced expression of apoptosis-related genes. Cytotoxicity experiment showed that the paclitaxel precursor, 10-deacetylbaccatin III, is cytotoxic to Sf9 cells, causing cell cycle arrest at the G2/M phase and disorder of the cytoskeleton. Metabolome analysis showed that heterologous expression of taxane genes in N. benthamiana affected the digestive system, steroid hormone and purine metabolism pathways of FAW larvae. In summary, this study provides a candidate approach for FAW control.

MsHDZ23, a Novel Miscanthus HD-ZIP Transcription Factor, Participates in Tolerance to Multiple Abiotic Stresses.

The homeodomain-leucine zipper (HD-ZIP) transcription factors, representing one of the largest plant-specific superfamilies, play important roles in the response to various abiotic stresses. However, the functional roles of HD-ZIPs in abiotic stress tolerance and the underlying mechanisms remain relatively limited in Miscanthus sinensis. In this study, we isolated an HD-ZIP TF gene, MsHDZ23, from Miscanthus and ectopically expressed it in Arabidopsis. Transcriptome and promoter analyses revealed that MsHDZ23 responded to salt, alkali, and drought treatments. The overexpression (OE) of MsHDZ23 in Arabidopsis conferred higher tolerance to salt and alkali stresses compared to wild-type (WT) plants. Moreover, MsHDZ23 was able to restore the hb7 mutant, the ortholog of MsHDZ23 in Arabidopsis, to the WT phenotype. Furthermore, MsHDZ23-OE lines exhibited significantly enhanced drought stress tolerance, as evidenced by higher survival rates and lower water loss rates compared to WT. The improved drought tolerance may be attributed to the significantly smaller stomatal aperture in MsHDZ23-OE lines compared to WT. Furthermore, the accumulation of the malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities in several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic plants. Collectively, these results demonstrate that MsHDZ23 functions as a multifunctional transcription factor in enhancing plant resistance to abiotic stresses.

ZmSMR10 Increases the Level of Endoreplication of Plants through Its Interactions with ZmPCNA2 and ZmCSN5B.

As a plant-specific endoreplication regulator, the SIAMESE-RELATED (SMR) family (a cyclin-dependent kinase inhibitor) plays an important role in plant growth and development and resistance to stress. Although the genes of the maize (Zea mays) SMR family have been studied extensively, the ZmSMR10 (Zm00001eb231280) gene has not been reported. In this study, the function of this gene was characterized by overexpression and silencing. Compared with the control, the transgenic plants exhibited the phenotypes of early maturation, dwarfing, and drought resistance. Expression of the protein in prokaryotes demonstrates that ZmSMR10 is a small protein, and the results of subcellular localization suggest that it travels functionally in the nucleus. Unlike ZmSMR4, yeast two-hybrid experiments demonstrated that ZmSMR10 does not interact strongly with with some cell cycle protein-dependent protein kinase (CDK) family members ZmCDKA;1/ZmCDKA;3/ZmCDKB1;1. Instead, it interacts strongly with ZmPCNA2 and ZmCSN5B. Based on these results, we concluded that ZmSMR10 is involved in the regulation of endoreplication through the interaction of ZmPCNA2 and ZmCSN5B. These findings provide a theoretical basis to understand the mechanism of the regulation of endoreplication and improve the yield of maize through the use of molecular techniques.

FvMYB108, a MYB Gene from Fragaria vesca, Positively Regulates Cold and Salt Tolerance of Arabidopsis.

MYB (myoblast) protein comes in large quantities and a wide variety of types and plays a role in most eukaryotes in the form of transcription factors (TFs). One of its important functions is to regulate plant responses to various stresses. However, the role of MYB TFs in regulating stress tolerance in strawberries is not yet well understood. Therefore, in order to investigate the response of MYB family members to abiotic stress in strawberries, a new MYB TF gene was cloned from Fragaria vesca (a diploid strawberry) and named FvMYB108 based on its structural characteristics and evolutionary relationships. After a bioinformatics analysis, it was determined that the gene belongs to the R2R3-MYB subfamily, and its conserved domain, phylogenetic relationships, predicted protein structure and physicochemical properties, subcellular localization, etc. were analyzed. After qPCR analysis of the expression level of FvMYB108 in organs, such as the roots, stems, and leaves of strawberries, it was found that this gene is more easily expressed in young leaves and roots. After multiple stress treatments, it was found that the target gene in young leaves and roots is more sensitive to low temperatures and salt stimulation. After these two stress treatments, various physiological and biochemical indicators related to stress in transgenic Arabidopsis showed corresponding changes, indicating that FvMYB108 may be involved in regulating the plant's ability to cope with cold and high-salt stress. Further research has found that the overexpression of this gene can upregulate the expression of AtCBF1, AtCOR47, AtERD10, and AtDREB1A related to low-temperature stress, as well as AtCCA1, AtRD29a, AtP5CS1, and AtSnRK2.4 related to salt stress, enhancing the ability of overexpressed plants to cope with stress.

Over-expression of the BnVIT-L2 gene improves the lateral root development and biofortification under iron stress.

The vacuolar iron transporter (VIT) family is responsible for absorbing and storing iron ions in vacuoles. Here, the BnVIT-L2 gene from Brassica napus has been cloned for the first time and was found to be expressed in multiple tissues and organs, induced by iron stress. The BnVIT-L2 protein is located in vacuolar membranes and has the ability to bind both iron and other bivalent metal ions. Over-expression of the BnVIT-L2 gene increased lateral root number and main root length, as well as chlorophyll and iron content in transgenic Arabidopsis plants (BnVIT-L2/At) exposed to iron stress, compared to wild type Col-0. Furthermore, over-expression of this gene improved the adaptability of transgenic B. napus plants (BnVIT-L2-OE) under iron stress. The regulation of plant tolerance under iron stress by BnVIT-L2 gene may involve in the signal of reactive oxygen species (ROS), as suggested by Ribosome profiling sequencing (Ribo-seq). This study provides a reference for investigating plant growth and biofortification under iron stress through the BnVIT-L2 gene.

CmoPIP1-4 confers drought tolerance in pumpkin by altering hydrogen sulfide signaling.

Drought is a major limiting factor for the growth and development of pumpkins. Plasma membrane intrinsic proteins (PIPs) are major water channels that play a crucial role in the regulation of cellular water status and solute trafficking during drought conditions. CmoPIP1-4 is a plasma membrane-localized protein that is significantly upregulated in roots and leaves under drought-stress conditions. In this study, the overexpression of CmoPIP1-4 enhances drought resistance in yeast. In contrast, CRISPR-mediated CmoPIP1-4 knockout in pumpkin roots increased drought sensitivity. This increased drought sensitivity of CmoPIP1-4 knockout plants is associated with a decline in the levels of hydrogen sulfide (H2S) and abscisic acid (ABA), accompanied by an increase in water loss caused by greater levels of transpiration and stomatal conductance. In addition, the sensitivity of CmoPIP1-4 CRISPR plants is further aggravated by reduced antioxidative enzyme activity, decreased proline and sugar contents, and extensive root damage. Furthermore, expression profiles of genes such as CmoHSP70s, CmoNCED3, CmoNCED4, and others involved in metabolic activities were markedly reduced in CmoPIP1-4 CRISPR plants. Moreover, we also discovered an interaction between the drought-responsive gene CmoDCD and CmoPIP1-4, indicating their potential role in activating H2S-mediated signaling in pumpkin, which could confer drought tolerance. The findings of our study collectively demonstrate CmoPIP1-4 plays a crucial role in the regulation of H2S-mediated signaling, influencing stomatal density and aperture in pumpkin plants, and thereby enhancing their drought tolerance.

Salicylic acid and jasmonic acid biosynthetic pathways are simultaneously activated in transgenic Arabidopsis expressing the rolB/C gene from Ipomoea batatas.

The Agrobacterium rhizogenes root oncogenic locus (rol) genes interfere with hormone balance by altering their synthesis and/or recognition, giving rise to varied impacts on the physiological characteristics of plants and cell cultures. The homolog of the rolB and rolC genes from Ipomoea batatas, named Ib-rolB/C, similarly induces morphological and physiological alterations in transgenic Arabidopsis thaliana; however, its role in plant hormonal homeostasis has not been previously defined. In this study, we found that external application of salicylic acid (SA) and methyl jasmonate (MeJA) significantly upregulated Ib-rolB/C in detached I. batatas leaves. Furthermore, heterologous expression of Ib-rolB/C in A. thaliana markedly enhanced the accumulation of SA and MeJA, and to a lesser extent, elevated abscisic acid (ABA) levels, through the modulation of genes specific to hormone biosynthesis. Even though the RolB/RolC homolog protein has a notable structural resemblance to the RolB protein from A. rhizogenes, it exhibits a distinct localization pattern, predominantly residing in the cytoplasm and certain discrete subcellular structures, instead of the nucleus. Consequently, the functions of RolB/RolC in both naturally and artificially transgenic plants are linked to changes in the hormonal state of the cells, though the underlying signaling pathways remain to be elucidated.

Ectopic expression of HaPEPC1 from the desert shrub Haloxylon ammodendron confers drought stress tolerance in Arabidopsis thaliana.

Phosphoenolpyruvate carboxylase (PEPC) plays a crucial role in the initial carbon fixation process in C4 plants. However, its nonphotosynthetic functions in Haloxylon ammodendron, a C4 perennial xerohalophytic shrub, are still poorly understood. Previous studies have reported the involvement of PEPC in plant responses to abiotic stresses such as drought and salt stress. However, the underlying mechanism of PEPC tolerance to drought stress has not been determined. In this study, we cloned the C4-type PEPC gene HaPEPC1 from H. ammodendron and investigated its biological function by generating transgenic Arabidopsis plants with ectopic expression of HaPEPC1. Our results showed that, compared with WT (wild-type) plants, ectopic expression of HaPEPC1 plants exhibited significantly greater germination rates and chlorophyll contents. Furthermore, under drought stress, the transgenic plants presented increased root length, fresh weight, photosynthetic capacity, and antioxidant enzyme activities, particularly ascorbate peroxidase and peroxidase. Additionally, the transgenic plants exhibited reduced levels of malondialdehyde, H2O2 (hydrogen peroxide), and O2- (superoxide radical). Transcriptome analysis indicated that ectopic expression of HaPEPC1 primarily regulated the expression of genes associated with the stress defence response, glutathione metabolism, and abscisic acid (ABA) synthesis and signalling pathways in response to drought stress. Taken together, these findings suggest that the ectopic expression of HaPEPC1 enhances the reduction of H2O2 and O2- in transgenic plants, thereby improving reactive oxygen species (ROS) scavenging capacity and enhancing drought tolerance. Therefore, the HaPEPC1 gene holds promise as a candidate gene for crop selection aimed at enhancing drought tolerance.

Comprehensive in silico characterization of NAC transcription factor family of Pinellia ternata and functional analysis of PtNAC66 under high-temperature tolerance in transgenic Arabidopsis thaliana.

Pinellia ternata, a valuable Chinese herb, suffers yield reduction due to "sprout tumble" under high temperatures. However, the mechanisms underlying its high-temperature stress remain poorly understood. NAM, ATAF1/2, and CUC2 (NAC) transcription factors regulate plant tissue growth and abiotic stress. Hence, there has been no comprehensive research conducted on NAC transcription factors in P. ternata. We identified 98 PtNAC genes unevenly distributed across 13 chromosomes, grouped into 15 families via phylogenetic analysis. Gene expression analysis revealed diverse expression patterns of PtNAC genes in different tissue types. Further studies revealed that PtNAC5/7/17/35/43/47/57/66/86 genes were highly expressed in various tissues of P. ternata and induced by heat stress, among which PtNAC66 was up-regulated at the highest folds induced by heat temperature. PtNAC66 is a nuclear protein that can selectively bind to the cis-responsive region NACRS but lacks the ability to activate transcription in yeast. For further research, PtNAC66 was cloned and transgenic Arabidopsis was obtained. PtNAC66 overexpression increased high-temperature tolerance compared to wild-type plants. Transcriptome profiling demonstrated that overexpression of PtNAC66 led to significant modification of genes responsible for regulating binding, catalytic activity, transcription regulator activity and transporter activity response genes. Additionally, PtNAC66 was found to bind the promoters of CYP707A3, MYB102 and NAC055, respectively, and inhibited their expression, affecting the high-temperature stress response in Arabidopsis. Our research established the foundation for functional studies of PtNAC genes in response to high-temperature forcing by characterizing the P. ternata NAC gene family and examining the biological role of PtNAC66 in plant high-temperature tolerance.

The RING zinc finger protein LbRZF1 promotes salt gland development and salt tolerance in Limonium bicolor.

The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named  RING  ZINC  FINGER PROTEIN  1 (LbRZF1). LbRZF1 was expressed in salt glands and in response to NaCl treatment. LbRZF1 showed no E3 ubiquitin ligase activity. The phenotypes of overexpression and knockout lines for LbRZF1 indicated that LbRZF1 positively regulated salt gland development and salt tolerance in L. bicolor. lbrzf1 mutants had fewer salt glands and secreted less salt than did the wild-type, whereas LbRZF1-overexpressing lines had opposite phenotypes, in keeping with the overall salt tolerance of these plants. A yeast two-hybrid screen revealed that LbRZF1 interacted with LbCATALASE2 (LbCAT2) and the transcription factor LbMYB113, leading to their stabilization. Silencing of LbCAT2 or LbMYB113 decreased salt gland density and salt tolerance. The heterologous expression of LbRZF1 in Arabidopsis thaliana conferred salt tolerance to this non-halophyte. We also identified the transcription factor LbMYB48 as an upstream regulator of LbRZF1 transcription. The study of LbRZF1 in the regulation network of salt gland development also provides a good foundation for transforming crops and improving their salt resistance.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrial outer membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.