PIN-FORMED Research Articles

Identification of Potato StPIN Gene Family and Regulation of Root Development by StPIN4.

The growth hormone export PIN-FORMED (PIN) is an important carrier for regulating the polar transport of plant growth hormones and plays an important role in plant growth and development. However, little is known about the characteristics and functions of PINs in potatoes. In this study, 10 PIN members were identified from potatoes and named StPIN1, StPIN2, StPIN3, StPIN4, StPIN5, StPIN6, StPIN7, StPIN8, StPIN9, and StPIN10 according to their positions in the potato chromosome In addition, the expression of 10 StPINs was analyzed by qRT-PCR during potato root development. The results showed that the StPIN4 gene plays an important regulatory role in potato root development, and its tissue expression varied greatly. Several cis-regulatory elements related to growth factors were also detected in the promoter region of the StPIN gene. The transgenic overexpressing StPIN4 in potato showed suppressed growth in root length and lateral root number, and StPIN4-interfering plants showed the opposite. These results suggested that StPIN4 plays a key role in the regulation of the potato root architecture.

Small peptide SiDVL/RTFLs from foxtail millet inhibit root growth through repressing auxin signaling in transgenic Arabidopsis.

SiDVLs inhibit auxin signaling to regulate root growth by enhancing the expression of Aux/IAAs and reducing the protein accumulation of PINs. The DEVIL/ ROTUNDIFOLIA (DVL/RTFL), a small polypeptide family, is conserved in seed plants and important in regulating plant growth and development. However, the molecular mechanisms remain largely unknown. Here, 27 SiDVLs were identified in foxtail millet genome. Overexpression of three SiDVLs in Arabidopsis (Arabidopsis thaliana) strongly repressed the plant growth, especially the root growth. We demonstrate that overexpression of SiDVLs enhances Auxin/Indole-3-Acetic Acids (Aux/IAAs) transcription, thereby weakening auxin signaling in the roots. Furthermore, SiDVLs reduced the protein levels of the auxin transporters PIN-formed 1 (PIN1), PIN2, and PIN7 in the roots. The impaired auxin signaling reduces the cell division and elongation. In conclusion, SiDVLs suppress cell division and elongation in root by inhibiting auxin signaling and transport, which lead to the reduced root growth.

Transport properties of canonical PIN-FORMED proteins from Arabidopsis and the role of the loop domain in auxin transport.

The phytohormone auxin is polarly transported in plants by PIN-FORMED (PIN) transporters and controls virtually all growth and developmental processes. Canonical PINs possess a long, largely disordered cytosolic loop. Auxin transport by canonical PINs is activated by loop phosphorylation by certain kinases. The structure of the PIN transmembrane domains was recently determined, their transport properties remained poorly characterized, and the role of the loop in the transport process was unclear. Here, we determined the quantitative kinetic parameters of auxin transport mediated by Arabidopsis PINs to mathematically model auxin distribution in roots and to test these predictions invivo. Using chimeras between transmembrane and loop domains of different PINs, we demonstrate a strong correlation between transport parameters and physiological output, indicating that the loop domain is not only required to activate PIN-mediated auxin transport, but it has an additional role in the transport process by a currently unknown mechanism.

Genome-wide gene network uncover temporal and spatial changes of genes in auxin homeostasis during fruit development in strawberry (F. × ananassa)

BackgroundThe plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (Fragaria vesca), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle.ResultsGenes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Heat Shock Transcription Factor and Heat Shock Proteins (HSF/HSP), APETALA2/Ethylene Responsive Factor (AP2/ERF) and MYB transcription factor groups.ConclusionsThese results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.

Polarly localized Bro1 domain proteins regulate PIN-FORMED abundance and root gravitropic growth in Arabidopsis

The developmental plasticity of the root system plays an essential role in the adaptation of plants to the environment. Among many other signals, auxin and its directional, intercellular transport are critical in regulating root growth and development. In particular, the PIN-FORMED2 (PIN2) auxin exporter acts as a key regulator of root gravitropic growth. Multiple regulators have been reported to be involved in PIN2-mediated root growth; however, our information remains incomplete. Here, we identified ROWY Bro1-domain proteins as important regulators of PIN2 sorting control. Genetic analysis revealed that Arabidopsis rowy1 single mutants and higher-order rowy1 rowy2 rowy3 triple mutants presented a wavy root growth phenotype. Cell biological experiments revealed that ROWY1 and PIN2 colocalized to the apical side of the plasma membrane in the root epidermis and that ROWYs are required for correct PM targeting of PIN2. In addition, ROWYs also affected PIN3 protein abundance in the stele, suggesting the potential involvement of additional PIN transporters as well as other proteins. A global transcriptome analysis revealed that ROWY genes are involved in the Fe2+ availability perception pathway. This work establishes ROWYs as important novel regulators of root gravitropic growth by connecting micronutrient availability to the proper subcellular targeting of PIN auxin transporters.

The SCOOP-MIK2 immune pathway modulates Arabidopsis root growth and development by regulating PIN-FORMED abundance and auxin transport.

Plants synthesize hundreds of small secretory peptides, which are perceived by the receptor-like kinase (RLK) family at the cell surface. Various signaling peptide-RLK pairs ensure plant adaptation to distinct environmental conditions. Here, we report that SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) immune peptides modulate root growth and development by regulating PIN-FORMED (PIN)-regulated polar auxin transport in Arabidopsis. The SCOOP4 and SCOOP12 treatments impaired root gravitropic growth, auxin redistribution in response to gravistimulation, and PIN abundance in the PM. Furthermore, genetic and cell biological analyses revealed that these physiological and cellular effects of SCOOP4 and SCOOP12 peptides are mediated by the receptor MALE DISCOVERER1-INTERACTING RECEPTOR LIKE KINASE2 (MIK2) and the downstream mitogen-activated kinase MPK6. Biochemical evidence indicates that MPK6 directly phosphorylates the cytosolic loop of PIN proteins. Our work established a link between the immune signaling peptide SCOOPs and root growth pathways, providing insights into the molecular mechanisms underlying plant root adaptive growth in the defense response.

Target of rapamycin coordinates auxin are involved in exogenous melatonin regulated low temperature tolerance in cucumber seedlings

Low temperature (LT) is an important environmental factor affecting the growth and yield of plants. Melatonin (MT) can effectively enhance the LT tolerance of cucumber. This study found that LT stress induced the expression of Comt1 (caffeic acid O-methyltransferase 1), with the highest expression being about 2-times that of the control. Meanwhile, the content of MT was found to be roughly 63.16% of that in the control samples. Compared with LT treatment alone, exogenous MT pretreatment upregulated the expression levels of TOR (Target of rapamycin), PIN1 (Pin-formed 1), and YUC4 (YUCCA 4), with maximum upregulations reaching approximately 66.67%, 79.32%, and 42.86%, respectively. These results suggest that MT may modulate the tolerance of cucumber seedlings to LT stress by regulating the expression of TOR, PIN1, and YUC4. In addition, co-treatment with AZD-8055 (a TOR inhibitor) or NPA (N-1-naphthylphthalamic acid, an auxin polar transport inhibitor) and MT attenuated MT-induced resistance to LT stress, leading to higher levels of reactive oxygen species (ROS), reduced antioxidant defense capacity, and increased damage to the membrane system in cucumber seedlings. Concurrently, the content of osmoregulatory substances and the photosynthesis decreased. These results demonstrate that both TOR and auxin were required for MT to alleviate LT-induced damage in cucumber. In summary, the present study demonstrates that TOR and auxin signaling synergistically contribute to alleviating LT damage in cucumber seedlings by exogenous MT. These findings help us understand the function of MT and provide insights into the regulatory network of MT that regulates the LT tolerance of plants.

Tradeoff between speed and robustness in primordium initiation mediated by auxin-CUC1 interaction

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates noise in auxin signaling that disrupts robustness in sepal initiation. Here, we find that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in drmy1 underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin noise in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for noisy auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases the intensity of auxin maxima and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we find that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Lastly, our model predicts that reducing global growth rate improves developmental robustness, which we validate experimentally. Thus, our study illustrates a tradeoff between speed and robustness during development.

Evolution of PIN gene family between monocotyledons and dicotyledons and VvPIN1 negatively regulates freezing tolerance in transgenic Arabidopsis.

The PIN-FORMED (PIN) proteins mediate the auxin flow throughout the plant and have been identified in many species. However, evolution differences in the PIN gene families have not been systematically analyzed, and their functions under abiotic stresses in grape are largely unexplored. In this study, 373 PIN genes were identified from 25 species and divided into 3 subgroups. Physicochemical properties analysis indicated that most of the PIN proteins were unstable alkaline hydrophobic proteins in nature. The synteny analysis showed that the PINs contained strong gene duplication. Motif composition revealed that PIN gene sequence differences between monocotyledons and dicotyledons were due to evolutionary-induced base loss, and the loss was more common in dicotyledonous. Meanwhile, the codon usage bias showed that the PINs showed stronger codon preference in monocotyledons, monocotyledons biased towards C3s and G3s, and dicotyledons biased towards A3s and T3s. In addition, the VvPIN1 can interact with VvCSN5. Significantly, under freezing treatment, the ion leakage, , H2O2, and malondialdehyde (MDA) were obviously increased, while the proline (Pro) content, peroxidase (POD) activity, and glutathione (GSH) content were decreased in VvPIN1-overexpressing Arabidopsis compared to the wild type (WT). And quantitative real-time PCR (qRT-PCR) showed that AtICE1, AtICE2, AtCBF1, AtCBF2, and AtCBF3 were down-regulated in overexpression lines. These results demonstrated that VvPIN1 negatively regulated the freezing tolerance in transgenic Arabidopsis. Collectively, this study provides a novel insight into the evolution and a basis for further studies on the biological functions of PIN genes in monocotyledons and dicotyledons.

Tradeoff Between Speed and Robustness in Primordium Initiation Mediated by Auxin-CUC1 Interaction.

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates noise in auxin signaling that disrupts robustness in sepal initiation. Here, we found that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in drmy1 underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin noise in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for noisy auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases auxin maxima intensity and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we found that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Lastly, our model predicts that reducing global growth rate improves developmental robustness, which we validated experimentally. Thus, our study illustrates a tradeoff between speed and robustness during development.

Auxin efflux carrier PsPIN4 identified through genome-wide analysis as vital factor of petal abscission.

PIN-FORMED (PIN) proteins, which function as efflux transporters, play many crucial roles in the polar transportation of auxin within plants. In this study, the exogenous applications of auxin IAA and TIBA were found to significantly prolong and shorten the florescence of tree peony (Paeonia suffruticosa Andr.) flowers. This finding suggests that auxin has some regulatory influence in petal senescence and abscission. Further analysis revealed a total of 8 PsPINs distributed across three chromosomes, which could be categorized into two classes based on phylogenetic and structural analysis. PsPIN1, PsPIN2a-b, and PsPIN4 were separated into the "long" PIN category, while PsPIN5, PsPIN6a-b, and PsPIN8 belonged to the "short" one. Additionally, the cis-regulatory elements of PsPIN promoters were associated with plant development, phytohormones, and environmental stress. These genes displayed tissue-specific expression, and phosphorylation sites were abundant throughout the protein family. Notably, PsPIN4 displayed distinct and elevated expression levels in roots, leaves, and flower organs. Expression patterns among the abscission zone (AZ) and adjacent areas during various flowering stages and IAA treatment indicate that PsPIN4 likely influences the initiation of peony petal abscission. The PsPIN4 protein was observed to be co-localized on both the plasma membrane and the cell nucleus. The ectopic expression of PsPIN4 reversed the premature flower organs abscission in the Atpin4 and significantly protracted florescence when introduced to Col Arabidopsis. Our findings established a strong basis for further investigation of PIN gene biological functions, particularly concerning intrinsic relationship between PIN-mediated auxin polar.

Cytosolic ABA Receptor Kinases phosphorylate the D6 PROTEIN KINASE leading to its stabilization which promotes Arabidopsis growth.

The polar auxin transport is required for proper plant growth and development. D6 PROTEIN KINASE (D6PK) is required for the phosphorylation of PIN-FORMED (PIN) auxin efflux carriers to regulate auxin transport, while the regulation of D6PK stabilization is still poorly understood. Here, we found that Cytosolic ABA Receptor Kinases (CARKs) redundantly interact with D6PK, and the interactions are dependent on CARKs' kinase activities. Similarly, CARK3 also could interact with paralogs of D6PK, including D6PKL1, D6PKL2, and D6PKL3. The genetic analysis shows that D6PK acts the downstream of CARKs to regulate Arabidopsis growth, including hypocotyl, leaf area, vein formation, and the length of silique. Loss-of-function of CARK3 in overexpressing GFP-D6PK plants leads to reduce the level of D6PK protein, thereby rescues plant growth. In addition, the cell-free degradation assays indicate that D6PK is degraded through 26 S proteasome pathway, while the phosphorylation by CARK3 represses this process in cells. In summary, D6PK stabilization by the CARK family is required for auxin-mediated plant growth and development.

Nonspecific phospholipases C3 and C4 interact with PIN-FORMED2 to regulate growth and tropic responses in Arabidopsis.

The dynamic changes in membrane phospholipids affect membrane biophysical properties and cell signaling, thereby influencing numerous biological processes. Nonspecific phospholipase C (NPC) enzymes hydrolyze common phospholipids to release diacylglycerol (DAG), which is converted to phosphatidic acid (PA) and other lipids. In this study, 2 Arabidopsis (Arabidopsis thaliana) tandemly arrayed genes, NPC3 and NPC4, were identified as critical factors modulating auxin-controlled plant growth and tropic responses. Moreover, NPC3 and NPC4 were shown to interact with the auxin efflux transporter PIN-FORMED2 (PIN2). The loss of NPC3 and NPC4 enhanced the endocytosis and vacuolar degradation of PIN2, which disrupted auxin gradients and slowed gravitropic and halotropic responses. Furthermore, auxin-triggered activation of NPC3 and NPC4 is required for the asymmetric PA distribution that controls PIN2 trafficking dynamics and auxin-dependent tropic responses. Collectively, our study reveals an NPC-derived PA signaling pathway in Arabidopsis auxin fluxes that is essential for fine-tuning the balance between root growth and environmental responses.

Transcriptome profiling of indole-3-butyric acid–induced adventitious root formation in softwood cuttings of walnut

Inducing adventitious root (AR) formation in mature walnut species (Juglans L.) is challenging. However, the AR formation of mature trees can be improved by rejuvenation. In rejuvenated cuttings, exogenous indole-3-butyric acid (IBA) is essential for AR formation, and the underlying mechanism is still not well understood. Therefore, we utilized transcriptome sequencing to investigate the mechanism of IBA-induced AR formation. Our results revealed that, in comparison to the control group, IBA treatment (9 mmol · L-1) significantly increased the endogenous indole-3-acetic acid (IAA) content, leading to an enhanced rooting rate. We performed RNA sequencing to identify differentially expressed genes (DEGs) between the IBA-treated and control (CK) groups at 1, 2, 3, and 5 days after cutting (DAC). The results showed that, compared to the control cuttings, there were 1 539, 889, 785, and 984 up-regulated genes and 2 791, 2 936, 3 017, and 1 752 down-regulated genes, at 1, 2, 3, and 5 DAC, respectively. Analysis of RNA-seq data revealed that G-type ATP-binding cassette 36/37 (ABCG36/37) and ATP-binding cassette subfamily D 1 (ABCD1), associated with IBA transport, were down-regulated in the rejuvenation cuttings. In contrast, PIN-FORMED (PIN) and PINOID (PID), associated with auxin efflux, were up-regulated. We identified 49 auxin/indole-3-acetic acid (AUX/IAA)-encoding genes, including IAA1, IAA3, IAA5, IAA6, IAA8, IAA11, IAA12, IAA19, and IAA20, which were up-regulated at 1–5 DAC in the rejuvenated cuttings. This study highlights that the overexpression of JrWOX5/11 in poplar significantly enhance AR growth, as evidenced by increased root length, surface area, volume, and quantity. Moreover, the co-expression network analysis involving JrWOX11 and JrWOX5 in walnut cuttings elucidates complex genetic interactions, underscoring their pivotal role in the formation of AR. Our data supported the following molecular mechanism of IBA-induced adventitious root formation. Firstly, IBA is converted to free IAA in peroxisomes. Then, the highly concentrated IAA in the procambium and parenchyma cells induces WUSCHEL-related homeobox 11 (WOX11) expression at two days. Finally, WOX11 acts redundantly to up-regulate WOX5, initiating the development of root primordia cells.

Genome Identification and Expression Profiling of the PIN-Formed Gene Family in Phoebe bournei under Abiotic Stresses.

PIN-formed (PIN) proteins-specific transcription factors that are widely distributed in plants-play a pivotal role in regulating polar auxin transport, thus influencing plant growth, development, and abiotic stress responses. Although the identification and functional validation of PIN genes have been extensively explored in various plant species, their understanding in woody plants-particularly the endangered species Phoebe bournei (Hemsl.) Yang-remains limited. P. bournei is an economically significant tree species that is endemic to southern China. For this study, we employed bioinformatics approaches to screen and identify 13 members of the PIN gene family in P. bournei. Through a phylogenetic analysis, we classified these genes into five sub-families: A, B, C, D, and E. Furthermore, we conducted a comprehensive analysis of the physicochemical properties, three-dimensional structures, conserved motifs, and gene structures of the PbPIN proteins. Our results demonstrate that all PbPIN genes consist of exons and introns, albeit with variations in their number and length, highlighting the conservation and evolutionary changes in PbPIN genes. The results of our collinearity analysis indicate that the expansion of the PbPIN gene family primarily occurred through segmental duplication. Additionally, by predicting cis-acting elements in their promoters, we inferred the potential involvement of PbPIN genes in plant hormone and abiotic stress responses. To investigate their expression patterns, we conducted a comprehensive expression profiling of PbPIN genes in different tissues. Notably, we observed differential expression levels of PbPINs across the various tissues. Moreover, we examined the expression profiles of five representative PbPIN genes under abiotic stress conditions, including heat, cold, salt, and drought stress. These experiments preliminarily verified their responsiveness and functional roles in mediating responses to abiotic stress. In summary, this study systematically analyzes the expression patterns of PIN genes and their response to abiotic stresses in P. bournei using whole-genome data. Our findings provide novel insights and valuable information for stress tolerance regulation in P. bournei. Moreover, the study offers significant contributions towards unraveling the functional characteristics of the PIN gene family.

Ectopic assembly of an auxin efflux control machinery shifts developmental trajectories.

Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.

Gibberellin promotes cambium reestablishment during secondary vascular tissue regeneration after girdling in an auxin-dependent manner in Populus.

Secondary vascular tissue (SVT) development and regeneration are regulated by phytohormones. In this study, we used an in vitro SVT regeneration system to demonstrate that gibberellin (GA) treatment significantly promotes auxin-induced cambium reestablishment. Altering GA content by overexpressing or knocking down ent-kaurene synthase (KS) affected secondary growth and SVT regeneration in poplar. The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE (PtoGAI) is expressed in a specific pattern during secondary growth and cambium regeneration after girdling. Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration, and the inhibition of cambium regeneration could be partially restored by GA application. Further analysis of the PtaDR5:GUS transgenic plants, the localization of PIN-FORMED 1 (PIN1) and the expression of auxin-related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1, which mediates auxin transport during SVT regeneration. Taken together, these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction.

Deficiency of Auxin Efflux Carrier OsPIN1b Impairs Chilling and Drought Tolerance in Rice

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 (O2−) 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.

BRIP1 and BRIP2 maintain root meristem by affecting auxin-mediated regulation.

This study reveals that mutations in BRIP1/2 subunits of the BAS complex disrupt root meristem development by decreasing PIN genes expression, affecting auxin transport, and downregulating essential root genes PLT. Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes play vital roles in plant development. BRAHMA-interacting proteins1 (BRIP1) and BRIP2 are subunits of BRAHMA (BRM)-associated SWI/SNF complex (BAS) in plants; however, their role and underlying regulatory mechanism in root development are still unknown. Here, we show that brip1 brip2 double mutants have a significantly shortened root meristem and an irregular arrangement in a portion of the root stem cell niche. The mutations in BRIP1 and BRIP2 cause decreased expression of the PIN-FORMED (PIN) genes, which in turn reduces the transport of auxin at the root tip, leading to the disruption of the accurate establishment of normal auxin concentration gradients in the stem cells. Chromatin immunoprecipitation (ChIP) experiments indicated that BRIP1 and BRIP2 directly bind to the PINs. Furthermore, we found a significant down-regulation in the expression of key root development genes, PLETHORA (PLT), in brip1 brip2. The brip1 brip2 plt1 plt2 quadruple mutations do not show further exacerbation in the short-root phenotype compared to plt1 plt2 double mutants. Using a dexamethasone (DEX)-inducible PLT2 transgenic line, we showed that acute overexpression of PLT2 partially rescues root meristem defects of brip1 brip2, suggesting that BRIP1 and BRIP2 act in part through the PLT1/2 pathway. Taken together, our results identify the critical role and the underlying mechanism of BRIP1/2 in maintaining the development of root meristem through the regulation of auxin output and expression of PLTs.

Suppression of pinoid mutant phenotypes by mutations in PIN-FORMED 1 and PIN1-GFP fusion.

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.