3,4-Dichlorophenylacetic acid acts as an auxin analog and induces beneficial effects in various crops.

Auxins and their analogs are widely used to promote root growth, flower and fruit development, and yield in crops. The action characteristics and application scope of various auxins are different. To overcome the limitations of existing auxins, expand the scope of applications, and reduce side effects, it is necessary to screen new auxin analogs. Here, we identified 3,4-dichlorophenylacetic acid (Dcaa) as having auxin-like activity and acting through the auxin signaling pathway in plants. At the physiological level, Dcaa promotes the elongation of oat coleoptile segments, the generation of adventitious roots, and the growth of crop roots. At the molecular level, Dcaa induces the expression of auxin-responsive genes and acts through auxin receptors. Molecular docking results showed that Dcaa can bind to auxin receptors, among which TIR1 has the highest binding activity. Application of Dcaa at the root tip of the DR5:GUS auxin-responsive reporter induces GUS expression in the root hair zone, which requires the PIN2 auxin efflux carrier. Dcaa also inhibits the endocytosis of PIN proteins like other auxins. These results provide a basis for the application of Dcaa in agricultural practices.

Toxicity Mechanisms of Nanoplastics on Crop Growth, Interference of Phyllosphere Microbes, and Evidence for Foliar Penetration and Translocation.

Despite the increasing prevalence of atmospheric nanoplastics (NPs), there remains limited research on their phytotoxicity, foliar absorption, and translocation in plants. In this study, we aimed to fill this knowledge gap by investigating the physiological effects of tomato leaves exposed to differently charged NPs and foliar absorption and translocation of NPs. We found that positively charged NPs caused more pronounced physiological effects, including growth inhibition, increased antioxidant enzyme activity, and altered gene expression and metabolite composition and even significantly changed the structure and composition of the phyllosphere microbial community. Also, differently charged NPs exhibited differential foliar absorption and translocation, with the positively charged NPs penetrating more into the leaves and dispersing uniformly within the mesophyll cells. Additionally, NPs absorbed by the leaves were able to translocate to the roots. These findings provide important insights into the interactions between atmospheric NPs and crop plants and demonstrate that NPs' accumulation in crops could negatively impact agricultural production and food safety.

AUX1, PIN3, and TAA1 collectively maintain fertility in Arabidopsis.

MAIN CONCLUSION: We found that auxin synthesis gene TAA1 and auxin polar transport genes AUX1 and PIN3 collectively maintain fertility and seed size in Arabidopsis. Auxin plays a vital role in plant gametophyte development and embryogenesis. The auxin synthesis gene TAA1 and the auxin polar transport genes AUX1 and PIN3 are expressed during Arabidopsis gametophyte and seed development. However, aux1, pin3, and taa1 single mutants only exhibit mild reproductive defects. We, therefore, generated aux1-T pin3 taa1-k2 and aux1-T pin3-2 taa1-k1 triple mutants by crossing or CRISPR/Cas9 technique. These triple mutants displayed severe reproductive defects with approximately 70% and 77%, respectively, of the siliques failing to elongate after anthesis. Reciprocal crosses and microscopy analyses showed that the development of pollen and ovules in the aux1 pin3 taa1 mutants was normal, whereas the filaments were remarkably short, which might be the cause of the silique sterility. Further analyses indicated that the development and morphology of aux1 pin3 taa1 seeds were normal, but their size was smaller compared with that of the wild type. These results indicate that AUX1, PIN3, and TAA1 act in concert to maintain fertility and seed size in Arabidopsis.

Biosynthesis- and transport-mediated dynamic auxin distribution during seed development controls seed size in Arabidopsis.

Auxin is indispensable to the fertilization-induced coordinated development of the embryo, endosperm, and seed coat. However, little attention has been given to the distribution pattern, maintenance mechanism, and function of auxin throughout the process of seed development. In the present study, we found that auxin response signals display a dynamic distribution pattern during Arabidopsis seed development. Shortly after fertilization, strong auxin response signals were observed at the funiculus, chalaza, and micropylar integument where the embryo attaches. Later, additional signals appeared at the middle layer of the inner integument (ii1') above the chalaza and the whole inner layer of the outer integument (oi1). These signals peaked when the seed was mature, then declined upon desiccation and disappeared in the dried seed. Auxin biosynthesis genes, including ASB1, TAA1, YUC1, YUC4, YUC8, and YUC9, contributed to the accumulation of auxin in the funiculus and seed coat. Auxin efflux carrier PIN3 and influx carrier AUX1 also contributed to the polar auxin distribution in the seed coat. PIN3 was expressed in the ii1 (innermost layer of the inner integument) and oi1 layers of the integument and showed polar localization. AUX1 was expressed in both layers of the outer integument and the endosperm and displayed a uniform localization. Further research demonstrated that the accumulation of auxin in the seed coat regulates seed size. Transgenic plants that specifically express the YUC8 gene in the oi2 or ii1 seed coat produced larger seeds. These results provide useful tools for cultivating high-yielding crops.

Transcription factors KNAT3 and KNAT4 are essential for integument and ovule formation in Arabidopsis.

Integuments form important protective cell layers surrounding the developing ovules in gymno- and angiosperms. Although several genes have been shown to influence the development of integuments, the transcriptional regulatory mechanism is still poorly understood. In this work, we report that the Class II KNOTTED1-LIKE HOMEOBOX (KNOX II) transcription factors KNOTTED1-LIKE HOMEBOX GENE 3 (KNAT3) and KNAT4 regulate integument development in Arabidopsis (Arabidopsis thaliana). KNAT3 and KNAT4 were co-expressed in inflorescences and especially in young developing ovules. The loss-of-function double mutant knat3 knat4 showed an infertility phenotype, in which both inner and outer integuments of the ovule are arrested at an early stage and form an amorphous structure as in the bell1 (bel1) mutant. The expression of chimeric KNAT3- and KNAT4-EAR motif repression domain (SRDX repressors) resulted in severe seed abortion. Protein-protein interaction assays demonstrated that KNAT3 and KNAT4 interact with each other and also with INNER NO OUTER (INO), a key transcription factor required for the outer integument formation. Transcriptome analysis showed that the expression of genes related with integument development is influenced in the knat3 knat4 mutant. The knat3 knat4 mutant also had a lower indole-3-acetic acid (IAA) content, and some auxin signaling pathway genes were downregulated. Moreover, transactivation analysis indicated that KNAT3/4 and INO activate the auxin signaling gene IAA INDUCIBLE 14 (IAA14). Taken together, our study identified KNAT3 and KNAT4 as key factors in integument development in Arabidopsis.

Nanotoxicological effects and transcriptome mechanisms of wheat (Triticum aestivum L.) under stress of polystyrene nanoplastics.

At present, the uptake and accumulation of nanoplastics by plants have raised particular concerns. However, molecular mechanisms underlying nanoplastic phytotoxicity are still vague and insufficient. To address this scientific gap, we analyzed the transcriptome response of hydroponically grown wheat (Triticum aestivum L.) to polystyrene nanoplastics (PSNPs) (100 nm) by integrating the differentially expressed gene analysis (DEGA) and the weighted gene correlation network analysis (WGCNA). PSNPs could significantly shape the gene expression patterns of wheat in a tissue-specific manner. Four candidate modules and corresponding hub genes associated with plant traits were identified using WGCNA. PSNPs significantly altered carbon metabolism, amino acid biosynthesis, mitogen-activated protein kinase (MAPK) signaling pathway-plant, plant hormone signal transduction, and plant-pathogen interaction Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, some Gene Ontology (GO) terms associated with the metal ion transport were further screened. These findings shed new light on the phytotoxic mechanism and environmental implication behind the interaction of nanoplastics and crop plants, and advance our understanding of the potential adverse effect induced by the presence of nanoplastics in agricultural systems.

The BAG2 and BAG6 Genes Are Involved in Multiple Abiotic Stress Tolerances in Arabidopsis Thaliana.

The BAG proteins are a family of multi-functional co-chaperones. In plants, BAG proteins were found to play roles both in abiotic and biotic stress tolerance. However, the function of Arabidopsis BAG2 remains largely unknown, whereas BAG6 is required for plants' defense to pathogens, although it remains unknown whether BAG6 is involved in plants' tolerance to abiotic stresses. Here, we show that both BAG2 and BAG6 are expressed in various tissues and are upregulated by salt, mannitol, and heat treatments and by stress-related hormones including ABA, ethylene, and SA. Germination of bag2, bag6 and bag2 bag6 seeds is less sensitive to ABA compared to the wild type (WT), whereas BAG2 and BAG6 overexpression lines are hypersensitive to ABA. bag2, bag6, and bag2 bag6 plants show higher survival rates than WT in drought treatment but display lower survival rates in heat-stress treatment. Consistently, these mutants showed differential expression of several stress- and ABA-related genes such as RD29A, RD29B, NCED3 and ABI4 compared to the WT. Furthermore, these mutants exhibit lower levels of ROS after drought and ABA treatment but higher ROS accumulation after heat treatment than the WT. These results suggest that BAG2 and BAG6 are negatively involved in drought stress but play a positive role in heat stress in Arabidopsis.

MES7 Modulates Seed Germination via Regulating Salicylic Acid Content in Arabidopsis.

Seed germination is an important phase transitional period of angiosperm plants during which seeds are highly sensitive to different environmental conditions. Although seed germination is under the regulation of salicylic acid (SA) and other hormones, the molecular mechanism underlying these regulations remains mysterious. In this study, we determined the expression of SA methyl esterase (MES) family genes during seed germination. We found that MES7 expression decreases significantly in imbibed seeds, and the dysfunction of MES7 decreases SA content. Furthermore, MES7 reduces and promotes seed germination under normal and salt stress conditions, respectively. The application of SA restores the seed germination deficiencies of mes7 mutants under different conditions. Taking together, our observations uncover a MeSA hydrolytic enzyme, MES7, regulates seed germination via altering SA titer under normal and abiotic stress conditions.

Mitochondrial heat-shock cognate protein 70 contributes to auxin-mediated embryo development.

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.

Membrane Sterol Composition in Arabidopsis thaliana Affects Root Elongation via Auxin Biosynthesis.

Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (ß-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.

UDP-Api/UDP-Xyl synthases affect plant development by controlling the content of UDP-Api to regulate the RG-II-borate complex.

Rhamnogalacturonan-II (RG-II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG-II molecules can form an RG-II-borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross-linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG-II dimer is still unclear. In this study we investigated the two homologous UDP-D-apiose/UDP-D-xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP-D-apiose (UDP-Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP-Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG-II-borate complex than wild-type Col-0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP-Api and RG-II-borate complex formation in plant growth and development.

A non-structural protein encoded by Rice Dwarf Virus targets to the nucleus and chloroplast and inhibits local RNA silencing.

RNA silencing is a potent antiviral mechanism in plants and animals. As a counter-defense, many viruses studied to date encode one or more viral suppressors of RNA silencing (VSR). In the latter case, how different VSRs encoded by a virus function in silencing remains to be fully understood. We previously showed that the nonstructural protein Pns10 of a Phytoreovirus, Rice dwarf virus (RDV), functions as a VSR. Here we present evidence that another nonstructural protein, Pns11, also functions as a VSR. While Pns10 was localized in the cytoplasm, Pns11 was localized both in the nucleus and chloroplasts. Pns11 has two bipartite nuclear localization signals (NLSs), which were required for nuclear as well as chloroplastic localization. The NLSs were also required for the silencing activities of Pns11. This is the first report that multiple VSRs encoded by a virus are localized in different subcellular compartments, and that a viral protein can be targeted to both the nucleus and chloroplast. These findings may have broad significance in studying the subcellular targeting of VSRs and other viral proteins in viral-host interactions.

AtXRN4 Affects the Turnover of Chosen miRNA*s in Arabidopsis.

Small RNA (sRNA) turnover is a key but poorly understood mechanism that determines the homeostasis of sRNAs. Animal XRN genes contribute the degradation of sRNAs, AtXRN2 and AtXRN3 also contribute the pri-miRNA processing and miRNA loop degradation in plants. However, the possible functions of the plant XRN genes in sRNA degradation are far from known. Here, we find that AtXRN4 contributes the turnover of plant sRNAs in Arabidopsis thaliana mainly by sRNA-seq, qRT-PCR and Northern blot. The mutation of AtXRN4 alters the sRNA profile and the accumulation of 21 nt sRNAs was increased. Some miRNA*s levels are significantly increased in xrn4 mutant plants. However, the accumulation of the primary miRNAs (pri-miRNAs) and miRNA precursors (pre-miRNAs) were generally unchanged in xrn4 mutant plants which indicates that AtXRN4 contributes the degradation of some miRNA*s. Moreover, AtXRN4 interacts with Arabidopsis Argonaute 2 (AtAGO2). This interaction takes place in Processing bodies (P-bodies). Taken together, our observations identified the interaction between XRN4 with AtAGO2 and suggested that plant XRN4 also contributes the turnover of sRNAs.

Sequestration of pyridinium herbicides in plants by carboxylated pillararenes possessing different alkyl chains.

We report that the sequestration of pyridinium-containing herbicides can be achieved on plant foliage through the strong supramolecular complexation with water-soluble pillararenes. The host-guest interaction appears to exert a protective effect on the plant growth, thus holding great promise in agricultural application.

The SMO1 Family of Sterol 4α-Methyl Oxidases Is Essential for Auxin- and Cytokinin-Regulated Embryogenesis.

In the plant sterol biosynthetic pathway, sterol 4α-methyl oxidase1 (SMO1) and SMO2 enzymes are involved in the removal of the first and second methyl groups at the C-4 position, respectively. SMO2s have been found to be essential for embryonic and postembryonic development, but the roles of SMO1s remain unclear. Here, we found that the three Arabidopsis (Arabidopsis thaliana) SMO1 genes displayed different expression patterns. Single smo1 mutants and smo1-1 smo1-3 double mutants showed no obvious phenotype, but the smo1-1 smo1-2 double mutant was embryo lethal. The smo1-1 smo1-2 embryos exhibited severe defects, including no cotyledon or shoot apical meristem formation, abnormal division of suspensor cells, and twin embryos. These defects were associated with enhanced and ectopic expression of auxin biosynthesis and response reporters. Consistently, the expression pattern and polar localization of PIN FORMED1, PIN FORMED7, and AUXIN RESISTANT1 auxin transporters were dramatically altered in smo1-1 smo1-2 embryos. Moreover, cytokinin biosynthesis and response were reduced in smo1-1 smo1-2 embryos. Tissue culture experiments further demonstrated that homeostasis between auxin and cytokinin was altered in smo1-1 smo1-2 heterozygous mutants. This disturbed balance of auxin and cytokinin in smo1-1 smo1-2 embryos was accompanied by unrestricted expression of the quiescent center marker WUSCHEL-RELATED HOMEOBOX5 Accordingly, exogenous application of either auxin biosynthesis inhibitor or cytokinin partially rescued the embryo lethality of smo1-1 smo1-2 Sterol analyses revealed that 4,4-dimethylsterols dramatically accumulated in smo1-1 smo1-2 heterozygous mutants. Together, these data demonstrate that SMO1s function through maintaining correct sterol composition to balance auxin and cytokinin activities during embryogenesis.

The Arabidopsis nucleoporin NUP1 is essential for megasporogenesis and early stages of pollen development.

KEY MESSAGE: Loss-of-function of nucleoporin NUP1 in Arabidopsis causes defect in both male and female gametogenesis. Its ovules are arrested during meiosis, and its pollen grains are aborted at mitosis I. Nuclear pore complex (NPC) plays crucial roles in nucleocytoplasmic trafficking of proteins and RNAs. The NPC contains approximately 30 different proteins termed nucleoporins (NUPs). So far, only a few of plant NUPs have been characterized. The Arabidopsis NUP1 was identified as an ortholog of the yeast NUP1 and animal NUP153. Loss-of-function of NUP1 in Arabidopsis caused fertility defect; however, the molecular mechanism of this defect remains unknown. Here, we found that both male and female gametogenesis of the nup1 mutants were defective. nup1 ovules were arrested from the meiosis stage onward; only approximately 6.7% and 3% ovules of the nup1-1 and nup1-4 mutants developed up to the FG7 stage, respectively. Pollen development of the nup1 mutants was arrested during the first mitotic division. In addition, enlarged pollen grains with increased DNA content were observed in the nup1 mutant. RNA-sequencing showed that expression levels of genes involved in pollen development or regulation of cell size were reduced dramatically in nup1 compared with wild type. These results suggest that NUP1 plays an important role in gametogenesis.

AUX1 acts upstream of PIN2 in regulating root gravitropism.

AUX1 and PIN2 auxin transporter are required for the asymmetric distribution of auxin for root gravitropic response. However, the relationship between AUX1 and PIN2 in root gravitropism is unclear. Here, we report that aux1-T mutant show stronger defects in root gravitropism than pin2-T, and aux1-T pin2-T double mutants display similar agravitropic phenotype to aux1-T. The gravity-induced asymmetric distribution of auxin responses could not be established in pin2-T, aux1-T and aux1-T pin2-T mutants; whereas aux1-T pin2-T double mutants showed similar auxin responses to aux1-T mutant. These findings support AUX1 plays a role in root gravitropism upstream of PIN2.

The Arabidopsis GPR1 Gene Negatively Affects Pollen Germination, Pollen Tube Growth, and Gametophyte Senescence.

Genes essential for gametophyte development and fertilization have been identified and studied in detail; however, genes that fine-tune these processes are largely unknown. Here, we characterized an unknown Arabidopsis gene, GTP-BINDING PROTEIN RELATED1 (GPR1). GPR1 is specifically expressed in ovule, pollen, and pollen tube. Enhanced green fluorescent protein-tagged GPR1 localizes to both nucleus and cytoplasm, and it also presents in punctate and ring-like structures. gpr1 mutants exhibit no defect in gametogenesis and seed setting, except that their pollen grains are pale in color. Scanning electron microscopy analyses revealed a normal patterned but thinner exine on gpr1 pollen surface. This may explain why gpr1 pollen grains are pale. We next examined whether GPR1 mutation affects post gametogenesis processes including pollen germination, pollen tube growth, and ovule senescence. We found that gpr1 pollen grains germinated earlier, and their pollen tubes elongated faster. Emasculation assay revealed that unfertilized gpr1 pistil expressed the senescence marker PBFN1:GUS (GUS: a reporter gene that encodes ß-glucuronidase) one-day earlier than the wild type pistil. Consistently, ovules and pollen grains of gpr1 mutants showed lower viability than those of the wild type at 4 to 5 days post anthesis. Together, these data suggest that GPR1 functions as a negative regulator of pollen germination, pollen tube growth, and gametophyte senescence to fine-tune the fertilization process.

Chronic mitochondrial calcium elevation suppresses leaf senescence.

Mitochondria Ca2+ overload has long been recognized as a cell death trigger. Unexpectedly, we demonstrated a signaling complex composed of Calmodulin (CaM), Arabidopsis thaliana Bcl-2-associated athanogene 5 (AtBAG5) and Heat-shock cognate 70 protein (Hsc70) within Arabidopsis thaliana mitochondria which transduces mitochondria Ca2+ elevations to suppress leaf senescence. Gain- and loss-of-function AtBAG5 mutant plants revealed that, mitochondria Ca2+ elevation significantly increase chlorophyll retention and decrease H2O2 level in dark-induced leaf senescence assay. Based on our findings, we proposed a molecular mechanism in which chronic mitochondria Ca2+ elevation reduced ROS levels and thus inhibits leaf senescence.

CaM/BAG5/Hsc70 signaling complex dynamically regulates leaf senescence.

Calcium signaling plays an essential role in plant cell physiology, and chaperone-mediated protein folding directly regulates plant programmed cell death. The Arabidopsis thaliana protein AtBAG5 (Bcl-2-associated athanogene 5) is unique in that it contains both a BAG domain capable of binding Hsc70 (Heat shock cognate protein 70) and a characteristic IQ motif that is specific for Ca(2+)-free CaM (Calmodulin) binding and hence acts as a hub linking calcium signaling and the chaperone system. Here, we determined crystal structures of AtBAG5 alone and in complex with Ca(2+)-free CaM. Structural and biochemical studies revealed that Ca(2+)-free CaM and Hsc70 bind AtBAG5 independently, whereas Ca(2+)-saturated CaM and Hsc70 bind AtBAG5 with negative cooperativity. Further in vivo studies confirmed that AtBAG5 localizes to mitochondria and that its overexpression leads to leaf senescence symptoms including decreased chlorophyll retention and massive ROS production in dark-induced plants. Mutants interfering the CaM/AtBAG5/Hsc70 complex formation leads to different phenotype of leaf senescence. Collectively, we propose that the CaM/AtBAG5/Hsc70 signaling complex plays an important role in regulating plant senescence.