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The crosstalk between clathrin-mediated endocytosis (CME) and the autophagy pathway has been reported in mammals; however, the interconnection of CME with autophagy has not been established in plants. Here, we report that the Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopies Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants in both autoimmunity and nutrient sensitivity. Accordingly, the autophagy pathway is significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, multiple assays demonstrate that CLC2 directly interacts with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP are subjected to autophagic degradation, and degradation of GFP-ATG8h is significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneous knockout of ATG8h and ATG8i by CRISPR-Cas9 results in enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results reveal a link between the function of CLCs and the autophagy pathway in Arabidopsis., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salicylic acid (SA) is a key phyto-hormone that is essential for plant immunity. SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1), a member of the CBP60 (CALMODULIN-BINDING PROTEIN60) gene family, is one of the major transcription factors regulating the expression of the genes in SA biosynthesis. SARD1 has been extensively studied in model plant Arabidopsis. However, the function of SARD1 homologues in SA biosynthesis and immune responses have rarely been investigated in other plant species. In this study, the CRISPR/CAS9 (Clustered Regularly Interspersed Short Palindromic Repeats/CAS9) technology was used in creating transgenic tobacco mutant lines with 6-8 alleles of four NtSARD1 homologous genes (NtSARD1a/1b/1c/1d) knocked out. No significant difference in morphological phenotype was observed between the transgenic knockout lines and the wild type tobacco plants, indicating that knocking out NtSARD1s does not affect the growth and development in tobacco. However, knocking out or partially knocking out of NtSARD1a/b/c/d resulted in a significantly reduced expression of NtICS1, the key gene in SA biosynthesis pathway, and thus the subsequently decreased SA/SAG accumulations in response to Pst DC3000 (Pseudomonas syrangae pv.tomato DC3000) infection, indicating a key role of NtSARD1 genes in SA biosynthesis in tobacco. As a consequence of reduced SA/SAG accumulation, the Pst DC3000-induced expression of NtPR genes as well as the resistance to Pst DC3000 were both significantly reduced in these knockout lines compared with the wild type tobacco plants. Interestingly, the reductions in the SA/SAG level, NtPR gene induction and Pst DC3000 resistance were positively correlated with the number of alleles being knocked out. Furthermore, LUC reporter gene driven by the promoter of NtICS1 containing two G(A/T)AATT(T/G) motifs could be activated by NtSARD1a, suggesting that NtSARD1a could bind to the core G(A/T)AATT(T/G) motifs and thus activate the expression of LUC reporter. Taken together, our results demonstrated that the NtSARD1 proteins play essential roles in SA biosynthesis and immune responses in tobacco. Our results also demonstrated that the CRISPR/CAS9 technology can overcome gene redundancy and is a powerful tool to study gene functions in polyploid plant species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

CBP60b (CALMODULIN-BINDING PROTEIN 60b) is a member of the CBP60 transcription factor family. In Arabidopsis, At CBP60b not only regulates growth and development but also activates the transcriptions in immune responses. So far, CBP60b has only been studied extensively in the model plant Arabidopsis and rarely in crops. In this study, Bean pod mottle virus (BPMV)-mediated gene silencing (BPMV-VIGS) was used to silence GmCBP60b.1/2 in soybean plants. The silencing of GmCBP60b.1/2 resulted in typical autoimmunity, such as dwarfism and enhanced resistance to both Soybean mosaic virus (SMV) and Pseudomonas syringae pv. glycinea ( Psg ). To further understand the roles of Gm CBP60b in immunity and circumvent the recalcitrance of soybean transformation, we generated transgenic tobacco lines that overexpress GmCBP60b.1 . The overexpression of GmCBP60b.1 also resulted in autoimmunity, including spontaneous cell death on the leaves, highly induced expression of PATHOGENESIS-RELATED ( PR ) genes, significantly elevated accumulation of defense hormone salicylic acid (SA), and significantly enhanced resistance to Pst DC3000 ( Pseudomonas syrangae pv. tomato DC3000). The transient coexpression of a luciferase reporter gene driven by the promoter of soybean SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 ( GmSARD1 ) ( ProGmSARD1::LUC ), together with GmCBP60b.1 driven by the 35S promoter, led to the activation of the LUC reporter gene, suggesting that Gm CBP60b.1 could bind to the core (A/T)AATT motifs within the promoter region of GmSARD1 and, thus, activate the expression of the LUC reporter. Taken together, our results indicate that Gm CBP60b.1/2 play both positive and negative regulatory roles in immune responses. These results also suggest that the function of CBP60b is conserved across plant species.

Receptor-like kinases (RLKs) are a large group of pattern recognition receptors (PRRs) and play a critical role in recognizing pathogens, transducing defense signals, and mediating the activation of immune defense responses. Although extensively studied in the model plant Arabidopsis, studies of RLKs in crops, including soybean, are limited. When a BAK1-interacting receptor-like kinase ( BIR1 ) homolog (referred to as GmBIR1 hereafter) was silenced by the BPMV ( Bean pod mottle virus )-induced gene silencing (BPMV-VIGS), it resulted in phenotypes that were reminiscent of constitutively activated defense responses, including a significantly stunted stature with observable cell death on the leaves of the silenced plants. In addition, both SA and H 2 O 2 were over-accumulated in the leaves of the GmBIR1 -silenced plants. Consistent with this autoimmune phenotype, GmBIR1 -silenced plants exhibited significantly enhanced resistance to both Pseudomonas syringae pv. glycinea ( Psg ) and Soybean mosaic virus (SMV), two different types of pathogens, compared to the vector control plants. Together, our results indicated that Gm BIR1 is a negative regulator of immunity in soybean and the function of BIR1 homologs is conserved in different plant species.

In animals, heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals perceived by numerous G protein-coupled receptors (GPCRs). However, no canonical GPCRs with guanine nucleotide exchange factor (GEF) activity are present in plant genomes. Accumulated evidence indicates that, instead of GPCRs, the receptor-like kinases (RLKs) function upstream of G proteins in plants. Regulator of G protein signaling 1 (RGS1) functions to convert the GTP-bound Gα to the GDP-bound form through its GTPase-accelerating protein (GAP) activity. Because of the intrinsic differences in the biochemical properties between Arabidopsis and animal Gα, the actions of animal and Arabidopsis RGS1 result in contrasting outcomes in G signaling activation/deactivation. Animal RGSs accelerate the deactivation of the activated G signaling, whereas Arabidopsis RGS1 prevents the activation of G signaling in the resting state. Phosphorylation of Arabidopsis RGS1 triggered by ligand-RLK recognition results in the endocytosis or degradation of RGS1, leading to the separation of RGS1 from Gα and thus the derepression of G signaling. Here, we summarize the involvement of the G proteins in plant immunity, with a special focus on the molecular mechanism of G signaling activation/deactivation regulated by RLKs and RGS1. We also provide a brief perspective on the outstanding questions that need to be addressed to fully understand G signaling in plant immunity., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

MAPK signaling pathways play critical roles in plant immunity. Here, we silenced multiple genes encoding MAPKs using virus-induced gene silencing mediated by Bean pod mottle virus to identify MAPK genes involved in soybean ( Glycine max ) immunity. Surprisingly, a strong hypersensitive response (HR) cell death was observed when soybean MAPK KINASE KINASE1 ( GmMEKK1 ), a homolog of Arabidopsis ( Arabidopsis thaliana ) MEKK1 , was silenced. The HR was accompanied by the overaccumulation of defense signaling molecules, salicylic acid (SA) and hydrogen peroxide. Genes involved in primary metabolism, translation/transcription, photosynthesis, and growth/development were down-regulated in GmMEKK1- silenced plants, while the expression of defense-related genes was activated. Accordingly, GmMEKK1 -silenced plants were more resistant to downy mildew ( Peronospora manshurica ) and Soybean mosaic virus compared with control plants. Silencing GmMEKK1 reduced the activation of GmMPK6 but enhanced the activation of Gm MPK3 in response to flg22 peptide. Unlike Arabidopsis MPK4, Gm MPK4 was not activated by either flg22 or SA. Interestingly, transient overexpression of GmMEKK1 in Nicotiana benthamiana also induced HR. Our results indicate that Gm MEKK1 plays both positive and negative roles in immunity and appears to differentially activate downstream MPKs by promoting Gm MPK6 activation but suppressing GmMPK3 activation in response to flg22. The involvement of Gm MPK4 kinase activity in cell death and in flg22- or SA-triggered defense responses in soybean requires further investigation., (© 2018 American Society of Plant Biologists. All rights reserved.)

Cross talk between phytohormones, nitric oxide (NO), and auxin has been implicated in the control of plant growth and development. Two recent reports indicate that NO promoted auxin signaling but inhibited auxin transport probably through S-nitrosylation. However, genetic evidence for the effect of S-nitrosylation on auxin physiology has been lacking. In this study, we used a genetic approach to understand the broader role of S-nitrosylation in auxin physiology in Arabidopsis. We compared auxin signaling and transport in Col-0 and gsnor1-3, a loss-of-function GSNOR1 mutant defective in protein de-nitrosylation. Our results showed that auxin signaling was impaired in the gsnor1-3 mutant as revealed by significantly reduced DR5-GUS/DR5-GFP accumulation and compromised degradation of AXR3NT-GUS, a useful reporter in interrogating auxin-mediated degradation of Aux/IAA by auxin receptors. In addition, polar auxin transport was compromised in gsnor1-3, which was correlated with universally reduced levels of PIN or GFP-PIN proteins in the roots of the mutant in a manner independent of transcription and 26S proteasome degradation. Our results suggest that S-nitrosylation and GSNOR1-mediated de-nitrosylation contribute to auxin physiology, and impaired auxin signaling and compromised auxin transport are responsible for the auxin-related morphological phenotypes displayed by the gsnor1-3 mutant., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY-YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY-YIG endonucleases. AtGRXS16-NTD was able to cleave λDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts.

Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 °C to 30 °C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 °C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.

While various signalling networks regulate plant responses to heat stress, the mechanisms regulating and unifying these diverse biological processes are largely unknown. Our previous studies indicate that the Arabidopsis monothiol glutaredoxin, AtGRXS17, is crucial for temperature-dependent postembryonic growth in Arabidopsis. In the present study, we further demonstrate that AtGRXS17 has conserved functions in anti-oxidative stress and thermotolerance in both yeast and plants. In yeast, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double-mutant cells to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm and the nuclear envelope but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. This enhanced thermotolerance correlated with increased catalase (CAT) enzyme activity and reduced H₂O₂ accumulation in AtGRXS17-expressing tomatoes. Furthermore, during heat stress, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing transgenic plants compared with wild-type controls. Thus, these findings suggest a specific protective role of a redox protein against temperature stress and provide a genetic engineering strategy to improve crop thermotolerance., (© 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Global environmental temperature changes threaten innumerable plant species. Although various signaling networks regulate plant responses to temperature fluctuations, the mechanisms unifying these diverse processes are largely unknown. Here, we demonstrate that an Arabidopsis monothiol glutaredoxin, AtGRXS17 (At4g04950), plays a critical role in redox homeostasis and hormone perception to mediate temperature-dependent postembryonic growth. AtGRXS17 expression was induced by elevated temperatures. Lines altered in AtGRXS17 expression were hypersensitive to elevated temperatures and phenocopied mutants altered in the perception of the phytohormone auxin. We show that auxin sensitivity and polar auxin transport were perturbed in these mutants, whereas auxin biosynthesis was not altered. In addition, atgrxs17 plants displayed phenotypes consistent with defects in proliferation and/or cell cycle control while accumulating higher levels of reactive oxygen species and cellular membrane damage under high temperature. Together, our findings provide a nexus between reactive oxygen species homeostasis, auxin signaling, and temperature responses.