Search Results (39)

Auxin plays a versatile role in regulating plant growth and development. The auxin efflux carrier PIN-FORMED (PIN) proteins dictate the distribution and maximum of auxin within various tissues. Despite extensive research on OsPINs in recent years, their functions in abiotic stress resistance, particularly cold tolerance, remain poorly understood. Here, we investigated the role of OsPIN5b in rice (Oryza sativa L.) growth and development, as well as its contribution to cold tolerance using overexpression technology. Overexpression of OsPIN5b (OE) resulted in reduced shoot height and a lower number of adventitious roots at the seedling stage. Transgenic rice plants exhibited an earlier heading date, stunted growth, and compromised agronomic traits, including shortened panicle length, decreased grain number per panicle, reduced seed size, and lower seed setting rate during the reproductive stage. Auxin content in the transgenic lines was significantly elevated, as indicated by the upregulation of the auxin-responsive gene OsIAA20 and increased auxin levels quantified using a newly developed method. Compared with wild-type plants, the cold tolerance of OE plants was markedly reduced, as evidenced by lower survival rates, higher levels of electrolyte leakage, and increased malondialdehyde (MDA) production following cold treatment. In line with this, the transgenic lines produced less soluble sugar and proline, while accumulating more hydrogen peroxide (H₂O₂) and superoxide anion radicals (${\mathrm{O}}_2^{-}$) after cold treatment. Furthermore, the activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), were notably decreased upon cold treatment compared with those in WT plants. Additionally, OsRBOHH, which plays a role in ROS production, was significantly upregulated in transgenic lines both before and after chilling stress, suggesting that OsRBOHH plays a potential role in regulating ROS production. Collectively, overexpression of OsPIN5b substantially disturbs auxin homeostasis, resulting in impaired plant architecture and agronomic traits. More importantly, the upregulation of OsPIN5b compromises rice cold tolerance by perturbing ROS homeostasis and adversely influencing the accumulation of soluble sugar and proline. Full article

Aluminum (Al) toxicity is a serious environmental constraint facing crop production in acidic soils, primarily due to the oxidative damage it causes to plant tissues. Alfalfa (Medicago sativa), a globally important forage crop, is highly susceptible to Al-induced stress, necessitating the development of Al-tolerant cultivars for sustainable forage production. In this study, we investigated the regulatory role of miR156 in Al stress response in alfalfa. Transcript analysis revealed significant downregulation of miR156 in alfalfa roots after 8 h of Al exposure, suggesting a negative role for miR156 in response to Al. To further investigate the role of miR156 in regulating agronomic traits and alfalfa’s Al tolerance, we utilized the short tandem target mimic (STTM) method to silence miR156 in alfalfa (MsSTTM156), which led to an upregulation of SQUAMOSA PROMOTER BINDING-LIKE (SPL) target genes, albeit with variable miR156 dose-dependent effects across different transgenic genotypes. Morphological characterization of MsSTTM156 plants revealed significant negative changes in root architecture, root and shoot biomass, as well as flowering time. Under Al stress, overexpression of miR156 in alfalfa (MsmiR156OE) resulted in stunted growth and reduced biomass, whereas moderate MsmiR156 silencing enhanced root dry weight and increased stem basal diameter. In contrast, MsmiR156OE reduced plant height, stem basal diameter, shoot branching, and overall biomass under Al stress conditions. At the molecular level, silencing miR156 modulated the transcription of cell wall-related genes linked to Al tolerance, such as polygalacturonase 1(MsPG1) and polygalacturonase 4 (MsPG4). Furthermore, miR156 influenced the expression of indole-3-acetic acid (IAA) transport-related genes auxin transporter-like protein (MsAUX1) and auxin efflux carrier components 2 (MsPIN2), with MsSTTM156 and MsmiR156OE plants showing lower and higher transcript levels, respectively, upon Al exposure. These findings reveal the multi-layered role of miR156 in mediating Al tolerance, providing valuable insights into the genetic strategies that regulate response to Al stress in alfalfa. Full article

Light provides seeds with information that is essential for the adjustment of their germination to the conditions that are most favorable for the successful establishment of the future seedling. The promotion of germination depends mainly on environmental factors, like temperature and light, as well as internal factors associated with the hormonal balance between gibberellins (GA) and abscisic acid (ABA), although other hormones such as auxins may act secondarily. While transcriptomic studies of light-germinating Arabidopsis thaliana seeds suggest that auxins and auxin transporters are necessary, there are still no functional studies connecting the activity of the auxin transporters in light-induced seed germination. In this study, we investigated the roles of two auxin efflux carrier (PIN3 and PIN7) proteins and one auxin influx (AUX1) carrier protein during Arabidopsis thaliana seed germination. By using next-generation sequencing (RNAseq), gene expression analyses, hormonal sensitivity assays, and the quantification of indole-3-acetic acid (IAA) levels, we assessed the functional roles of PIN3, PIN7, and AUX1 during light-induced seed germination. We showed that auxin levels are increased 24 h after a red-pulse (Rp). Additionally, we evaluated the germination responses of pin3, pin7, and aux1 mutant seeds and showed that PIN3, PIN7, and AUX1 auxin carriers are important players in the regulation of seed germination. By using gene expression analysis in water, fluridone (F), and ABA+F treated seeds, we confirmed that Rp-induced seed germination is associated with auxin transport, and ABA controls the function of PIN3, PIN7, and AUX1 during this process. Overall, our results highlight the relevant and positive role of auxin transporters in germinating the seeds of Arabidopsis thaliana. Full article

Significant progress has been made in the functions of auxin efflux transporter PIN-FORMED (PIN) genes for the regulation of growth and development in rice. However, knowledge on the roles of OsPIN genes in abiotic stresses is limited. We previously reported that the mutation of OsPIN1b alters rice architecture and root gravitropism, while the role of OsPIN1b in the regulation of rice abiotic stress adaptations is still largely elusive. In the present study, two homozygous ospin1b mutants (C1b-1 and C1b-2) were employed to investigate the roles of OsPIN1b in regulating abiotic stress adaptations. Low temperature gradually suppressed OsPIN1b expression, while osmotic stress treatment firstly induced and then inhibited OsPIN1b expression. Most OsPIN genes and auxin biosynthesis key genes OsYUC were up-regulated in ospin1b leaves, implying that auxin homeostasis is probably disturbed in ospin1b mutants. The loss of function of OsPIN1b significantly decreased rice chilling tolerance, which was evidenced by decreased survival rate, increased death cells and ion leakage under chilling conditions. Compared with the wild-type (WT), ospin1b mutants accumulated more hydrogen peroxide (H₂O₂) and less superoxide anion radicals $\left({\mathrm{O}}_2^{-}\right)$ after chilling treatment, indicating that reactive oxygen species (ROS) homeostasis is disrupted in ospin1b mutants. Consistently, C-repeat binding factor (CBF)/dehydration-responsive element binding factor (DREB) genes were downregulated in ospin1b mutants, implying that OsDREB genes are implicated in OsPIN1b-mediated chilling impairment. Additionally, the mutation of OsPIN1b led to decreased sensitivity to abscisic acid (ABA) treatment in seed germination, impaired drought tolerance in the seedlings and changed expression of ABA-associated genes in rice roots. Taken together, our investigations revealed that OsPIN1b is implicated in chilling and drought tolerance in rice and provide new insight for improving abiotic stress tolerance in rice. Full article

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore. Full article

Auxins are responsible for a large part of the plant development process. To exert their action, they must move throughout the plant and from cell to cell, which is why plants have developed complex transport systems for indole-3-acetic acid (IAA). These transporters involve proteins that transport IAA into cells, transporters that move IAA to or from different organelles, mainly the endoplasmic reticulum, and transporters that move IAA out of the cell. This research determined that Persea americana has 12 PIN transporters in its genome. The twelve transporters are expressed during different stages of development in P. americana zygotic embryos. Using different bioinformatics tools, we determined the type of transporter of each of the P. americana PIN proteins and their structure and possible location in the cell. We also predict the potential phosphorylation sites for each of the twelve-PIN proteins. The data show the presence of highly conserved sites for phosphorylation and those sites involved in the interaction with the IAA. Full article

In de novo shoot organogenesis (DNSO) plant cells develop into new shoots, without the need of an existing meristem. Generally, this process is triggered by wounding and specific growth regulators, such as auxins and cytokinins. Despite the potential significance of the plant hormone ethylene in DNSO, its effect in regeneration processes of woody species has not been thoroughly investigated. To address this gap, Solanum betaceum Cav. was used as an experimental model to explore the role of this hormone on DNSO and potentially extend the findings to other woody species. In this work it was shown that ethylene positively regulates DNSO from tamarillo leaf explants. Ethylene precursors ACC and ethephon stimulated shoot regeneration by increasing the number of buds and shoots regenerated. In contrast, the inhibition of ethylene biosynthesis or perception by AVG and AgNO₃ decreased shoot regeneration. Organogenic callus induced in the presence of ethylene precursors showed an upregulated expression of the auxin efflux carrier gene PIN1, suggesting that ethylene may enhance shoot regeneration by affecting auxin distribution prior to shoot development. Additionally, it was found that the de novo shoot meristems induced in explants in which ethylene biosynthesis and perception was suppressed were unable to further develop into elongated shoots. Overall, these results imply that altering ethylene levels and perception could enhance shoot regeneration efficiency in tamarillo. Moreover, we offer insights into the possible molecular mechanisms involved in ethylene-induced shoot regeneration. Full article

Phytopathogens are well known for their devastating activity that causes worldwide significant crop losses. However, their exploitation for crop welfare is relatively unknown. Here, we show that the microbial volatile organic compound (mVOC) profile of the bacterial phytopathogen, Erwinia amylovora, enhances Arabidopsis thaliana shoot and root growth. GC-MS head-space analyses revealed the presence of typical microbial volatiles, including 1-nonanol and 1-dodecanol. E. amylovora mVOCs triggered early signaling events including plasma transmembrane potential Vm depolarization, cytosolic Ca²⁺ fluctuation, K⁺-gated channel activity, and reactive oxygen species (ROS) and nitric oxide (NO) burst from few minutes to 16 h upon exposure. These early events were followed by the modulation of the expression of genes involved in plant growth and defense responses and responsive to phytohormones, including abscisic acid, gibberellin, and auxin (including the efflux carriers PIN1 and PIN3). When tested, synthetic 1-nonanol and 1-dodecanol induced root growth and modulated genes coding for ROS. Our results show that E. amylovora mVOCs affect A. thaliana growth through a cascade of early and late signaling events that involve phytohormones and ROS. Full article

Nitric oxide (NO) is a versatile signal molecule that mediates environmental and hormonal signals orchestrating plant development. NO may act via reversible S-nitrosation of proteins during which an NO moiety is added to a cysteine thiol to form an S-nitrosothiol. In plants, several proteins implicated in hormonal signaling have been reported to undergo S-nitrosation. Here, we report that the Arabidopsis ROP2 GTPase is a further potential target of NO-mediated regulation. The ROP2 GTPase was found to be required for the root shortening effect of NO. NO inhibits primary root growth by altering the abundance and distribution of the PIN1 auxin efflux carrier protein and lowering the accumulation of auxin in the root meristem. In rop2-1 insertion mutants, however, wild-type-like root size of the NO-treated roots were maintained in agreement with wild-type-like PIN1 abundance in the meristem. The ROP2 GTPase was shown to be S-nitrosated in vitro, suggesting that NO might directly regulate the GTPase. The potential mechanisms of NO-mediated ROP2 GTPase regulation and ROP2-mediated NO signaling in the primary root meristem are discussed. Full article

Mikania micrantha, recognized as one of the world’s top 10 pernicious weeds, is a rapidly spreading tropical vine that has invaded the coastal areas of South China, causing serious economic losses and environmental damage. Rapid stem growth is an important feature of M. micrantha which may be related to its greater number of genes involved in auxin signaling and transport pathways and its ability to synthesize more auxin under adverse conditions to promote or maintain stem growth. Plant growth and development is closely connected to the regulation of endogenous hormones, especially the polar transport and asymmetric distribution of auxin. The PIN-FORMED (PIN) auxin efflux carrier gene family plays a key role in the polar transport of auxin and then regulates the growth of different plant tissues, which could indicate that the rapid growth of M. micrantha is closely related to this PIN-dependent auxin regulation. In this study, 11 PIN genes were identified and the phylogenetic relationship and structural compositions of the gene family in M. micrantha were analyzed by employing multiple bioinformatic methods. The phylogenetic analysis indicated that the PIN proteins could be divided into five distinct clades. The structural analysis revealed that three putative types of PIN (canonical, noncanonical and semi-canonical) exist among the proteins according to the length and the composition of the hydrophilic domain. The majority of the PINs were involved in the process of axillary bud differentiation and stem response under abiotic stress, indicating that M. micrantha may regulate its growth, development and stress response by regulating PIN expression in the axillary bud and stem, which may help explain its strong growth ability and environmental adaptability. Our study emphasized the structural features and stress response patterns of the PIN gene family and provided useful insights for further study into the molecular mechanism of auxin-regulated growth and control in M. micrantha. Full article

The distribution and content of auxin within plant tissues affect a variety of important growth and developmental processes. Polar auxin transport (PAT), mainly mediated by auxin influx and efflux transporters, plays a vital role in determining auxin maxima and gradients in plants. The auxin efflux carrier PIN-FORMED (PIN) family is one of the major protein families involved in PAT. Rice (Oryza sativa L.) genome possesses 12 OsPIN genes. However, the detailed functions of OsPIN genes involved in regulating the rice architecture and gravity response are less well understood. In the present study, OsPIN1b was disrupted by CRISPR/Cas9 technology, and its roles in modulating rice architecture and root gravitropism were investigated. Tissue-specific analysis showed that OsPIN1b was mainly expressed in roots, stems and sheaths at the seedling stage, and the transcript abundance was progressively decreased during the seedling stages. Expression of OsPIN1b could be quickly and greatly induced by NAA, indicating that OsPIN1b played a vital role in PAT. IAA homeostasis was disturbed in ospin1b mutants, as evidenced by the changed sensitivity of shoot and root to NAA and NPA treatment, respectively. Mutation of OsPIN1b resulted in pleiotropic phenotypes, including decreased growth of shoots and primary roots, reduced adventitious root number in rice seedlings, as well as shorter and narrower leaves, increased leaf angle, more tiller number and decreased plant height and panicle length at the late developmental stage. Moreover, ospin1b mutants displayed a curly root phenotype cultured with tap water regardless of lighting conditions, while nutrient solution culture could partially rescue the curly root phenotype in light and almost completely abolish this phenotype in darkness, indicating the involvement of the integration of light and nutrient signals in root gravitropism regulation. Additionally, amyloplast sedimentation was impaired in the peripheral tiers of the ospin1b root cap columella cell, while it was not the main contributor to the abnormal root gravitropism. These data suggest that OsPIN1b not only plays a vital role in regulating rice architecture but also functions in regulating root gravitropism by the integration of light and nutrient signals. Full article

Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors. Full article

Molecular responses of plants to natural phytotoxins comprise more general and compound-specific mechanisms. How phytotoxic chalcones and other flavonoids inhibit seedling growth was widely studied, but how they interfere with seed germination is largely unknown. The dihydrochalcone and putative allelochemical myrigalone A (MyA) inhibits seed germination and seedling growth. Transcriptome (RNAseq) and hormone analyses of Lepidium sativum seed responses to MyA were compared to other bioactive and inactive compounds. MyA treatment of imbibed seeds triggered the phased induction of a detoxification programme, altered gibberellin, cis-(+)-12-oxophytodienoic acid and jasmonate metabolism, and affected the expression of hormone transporter genes. The MyA-mediated inhibition involved interference with the antioxidant system, oxidative signalling, aquaporins and water uptake, but not uncoupling of oxidative phosphorylation or p-hydroxyphenylpyruvate dioxygenase expression/activity. MyA specifically affected the expression of auxin-related signalling genes, and various transporter genes, including for auxin transport (PIN7, ABCG37, ABCG4, WAT1). Responses to auxin-specific inhibitors further supported the conclusion that MyA interferes with auxin homeostasis during seed germination. Comparative analysis of MyA and other phytotoxins revealed differences in the specific regulatory mechanisms and auxin transporter genes targeted to interfere with auxin homestasis. We conclude that MyA exerts its phytotoxic activity by multiple auxin-dependent and independent molecular mechanisms. Full article

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins. Full article

Novel molecular pinball machines of the plasma membrane control cytosolic Ca²⁺ levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca²⁺ channels; 4. auxin-efflux “PIN” proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants, “proton pinballs” eject Ca²⁺ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca²⁺. Freed Ca²⁺ ions flow down the electrostatic gradient through open Ca²⁺ channels into the cytosol, thus activating numerous Ca²⁺-dependent activities. Clearly, cytosolic Ca²⁺ levels depend on the activity of the proton pump, the state of Ca²⁺ channels and the size of the periplasmic AGP-Ca²⁺ capacitor; proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin. Auxin efflux carriers conveniently known as “PIN” proteins (null mutants are pin-shaped) pump auxin from cell to cell. Mechanosensitive Ca²⁺ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca²⁺. Cell expansion also triggers proton pump/pinball activity by the mechanotransduction of wall stress via Hechtian adhesion, thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth. Finally, the Ca²⁺ homeostasis of plants depends on cell surface external storage as a source of dynamic Ca²⁺, unlike the internal ER storage source of animals, where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarizes a sixty-year Odyssey. Full article