Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Long extrachromosomal circular DNA identification by fusing sequence-derived features of physicochemical properties and nucleotide distribution patterns.

Long extrachromosomal circular DNA (leccDNA) regulates several biological processes such as genomic instability, gene amplification, and oncogenesis. The identification of leccDNA holds significant importance to investigate its potential associations with cancer, autoimmune, cardiovascular, and neurological diseases. In addition, understanding these associations can provide valuable insights about disease mechanisms and potential therapeutic approaches. Conventionally, wet lab-based methods are utilized to identify leccDNA, which are hindered by the need for prior knowledge, and resource-intensive processes, potentially limiting their broader applicability. To empower the process of leccDNA identification across multiple species, the paper in hand presents the very first computational predictor. The proposed iLEC-DNA predictor makes use of SVM classifier along with sequence-derived nucleotide distribution patterns and physicochemical properties-based features. In addition, the study introduces a set of 12 benchmark leccDNA datasets related to three species, namely Homo sapiens (HM), Arabidopsis Thaliana (AT), and Saccharomyces cerevisiae (SC/YS). It performs large-scale experimentation across 12 benchmark datasets under different experimental settings using the proposed predictor, more than 140 baseline predictors, and 858 encoder ensembles. The proposed predictor outperforms baseline predictors and encoder ensembles across diverse leccDNA datasets by producing average performance values of 81.09%, 62.2% and 81.08% in terms of ACC, MCC and AUC-ROC across all the datasets. The source code of the proposed and baseline predictors is available at https://github.com/FAhtisham/Extrachrosmosomal-DNA-Prediction . To facilitate the scientific community, a web application for leccDNA identification is available at https://sds_genetic_analysis.opendfki.de/iLEC_DNA/.

Metal-Organic Framework-Mediated Delivery of Nucleic Acid across Intact Plant Cells.

Plant synthetic biology is applied in sustainable agriculture, clean energy, and biopharmaceuticals, addressing crop improvement, pest resistance, and plant-based vaccine production by introducing exogenous genes into plants. This technique faces challenges delivering genes due to plant cell walls and intact cell membranes. Novel approaches are required to address this challenge, such as utilizing nanomaterials known for their efficiency and biocompatibility in gene delivery. This work investigates metal-organic frameworks (MOFs) for gene delivery in intact plant cells by infiltration. Hence, small-sized ZIF-8 nanoparticles (below 20 nm) were synthesized and demonstrated effective DNA/RNA delivery into Nicotiana benthamiana leaves and Arabidopsis thaliana roots, presenting a promising and simplified method for gene delivery in intact plant cells. We further demonstrate that small-sized ZIF-8 nanoparticles protect RNA from RNase degradation and successfully silence an endogenous gene by delivering siRNA in N. benthamiana leaves.

Lineage-specific gene duplication and expansion of DUF1216 gene family in Brassicaceae.

Proteins containing domain of unknown function (DUF) are prevalent in eukaryotic genome. The DUF1216 proteins possess a conserved DUF1216 domain resembling to the mediator protein of Arabidopsis RNA polymerase II transcriptional subunit-like protein. The DUF1216 family are specifically existed in Brassicaceae, however, no comprehensive evolutionary analysis of DUF1216 genes have been performed. We performed a first comprehensive genome-wide analysis of DUF1216 proteins in Brassicaceae. Totally 284 DUF1216 genes were identified in 27 Brassicaceae species and classified into four subfamilies on the basis of phylogenetic analysis. The analysis of gene structure and conserved motifs revealed that DUF1216 genes within the same subfamily exhibited similar intron/exon patterns and motif composition. The majority members of DUF1216 genes contain a signal peptide in the N-terminal, and the ninth position of the signal peptide in most DUF1216 is cysteine. Synteny analysis revealed that segmental duplication is a major mechanism for expanding of DUF1216 genes in Brassica oleracea, Brassica juncea, Brassica napus, Lepidium meyneii, and Brassica carinata, while in Arabidopsis thaliana and Capsella rubella, tandem duplication plays a major role in the expansion of the DUF1216 gene family. The analysis of Ka/Ks (non-synonymous substitution rate/synonymous substitution rate) ratios for DUF1216 paralogous indicated that most of gene pairs underwent purifying selection. DUF1216 genes displayed a specifically high expression in reproductive tissues in most Brassicaceae species, while its expression in Brassica juncea was specifically high in root. Our studies offered new insights into the phylogenetic relationships, gene structures and expressional patterns of DUF1216 members in Brassicaceae, which provides a foundation for future functional analysis.

Three new discovery effector proteins from Candidatus Liberibacter asiaticus psy62 inhibit plant defense through interaction with AtCAT3 and AtGAPA.

KEY MESSAGE: We identify three SDEs that inhibiting host defence from Candidatus Liberibacter asiaticus psy62, which is an important supplement to the pathogenesis of HLB. Candidatus Liberibacter asiaticus (CLas) is the main pathogen of citrus Huanglongbing (HLB). 38 new possible sec-dependent effectors (SDEs) of CLas psy62 were predicted by updated predictor SignalP 5.0, which 12 new SDEs were found using alkaline phosphate assay. Among them, SDE4310, SDE4435 and SDE4955 inhibited hypersensitivity reactions (HR) in Arabidopsis thaliana (Arabidopsis, At) and Nicotiana benthamiana leaves induced by pathogens, which lead to a decrease in cell death and reactive oxygen species (ROS) accumulation. And the expression levels of SDE4310, SDE4435, and SDE4955 genes elevated significantly in mild symptom citrus leaves. When SDE4310, SDE4435 and SDE4955 were overexpressed in Arabidopsis, HR pathway key genes pathogenesis-related 2 (PR2), PR5, nonexpressor of pathogenesis-related 1 (NPR1) and isochorismate synthase 1 (ICS1) expression significantly decreased and the growth of pathogen was greatly increased relative to control with Pst DC3000/AvrRps4 treatment. Our findings also indicated that SDE4310, SDE4435 and SDE4955 interacted with AtCAT3 (catalase 3) and AtGAPA (glyceraldehyde-3-phosphate dehydrogenase A). In conclusion, our results suggest that SDE4310, SDE4435 and SDE4955 are CLas psy62 effector proteins that may have redundant functions. They inhibit ROS burst and cell death by interacting with AtCAT3 and AtGAPA to negatively regulate host defense.

Identification and Expression Analysis of R2R3-MYB Transcription Factors Associated with Flavonoid Biosynthesis in Panax quinquefolius.

Panax quinquefolius L. is an important medicinal plant, and flavonoids are among its main secondary metabolites. The R2R3-MYB transcription factor plays an irreplaceable role in plant growth, development, and secondary metabolism. In our study, we identified 159 R2R3-MYBs and analyzed their physical and chemical properties in P. quinquefolius. The protein length of 159 PqMYBs varied from 107 to 1050 amino acids. The molecular weight ranged from 12.21 to 116.44 kDa. The isoelectric point was between 4.57 and 10.34. We constructed a phylogenetic tree of P. quinquefolius and Arabidopsis thaliana R2R3-MYB family members, and PqMYB members were divided into 33 subgroups. Transcriptome data analysis showed that the expression patterns of PqMYBs in root, leaf, and flower were significantly different. Following the MeJA treatment of seedlings, five candidate PqMYB genes demonstrated a response. A correlation analysis of PqMYBs and candidate flavonoid pathway genes showed that PqMYB2, PqMYB46, and PqMYB72 had correlation coefficients that were higher than 0.8 with PqCHS, PqANS4, and PqCCoAMT10, respectively. Furthermore, a transient expression assay confirmed that the three PqMYBs were localized in the nucleus. We speculated that these three PqMYBs were related to flavonoid biosynthesis in P. quinquefolius. These results provided a theoretical basis and a new perspective for further understanding the R2R3-MYB gene family and the biosynthesis mechanism of secondary metabolites in P. quinquefolius.

Identification of MATE Family and Characterization of GmMATE13 and GmMATE75 in Soybean's Response to Aluminum Stress.

The multidrug and toxic compound extrusion (MATE) proteins are coding by a secondary transporter gene family, and have been identified to participate in the modulation of organic acid exudation for aluminum (Al) resistance. The soybean variety Glycine max "Tamba" (TBS) exhibits high Al tolerance. The expression patterns of MATE genes in response to Al stress in TBS and their specific functions in the context of Al stress remain elusive. In this study, 124 MATE genes were identified from the soybean genome. The RNA-Seq results revealed significant upregulation of GmMATE13 and GmMATE75 in TBS upon exposure to high-dose Al3+ treatment and both genes demonstrated sequence homology to citrate transporters of other plants. Subcellular localization showed that both proteins were located in the cell membrane. Transgenic complementation experiments of Arabidopsis mutants, atmate, with GmMATE13 or GmMATE75 genes enhanced the Al tolerance of the plant due to citrate secretion. Taken together, this study identified GmMATE13 and GmMATE75 as citrate transporter genes in TBS, which could improve citrate secretion and enhance Al tolerance. Our findings provide genetic resources for the development of plant varieties that are resistant to Al toxicity.

TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns.

BACKGROUND: Tonoplast intrinsic proteins (TIPs), which typically mediate water transport across vacuolar membranes, play an essential role in plant growth, development, and stress responses. However, their characterization in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant of the Cyperaceae family, is still in the infancy. RESULTS: In this study, a first genome-wide characterization of the TIP subfamily was conducted in tigernut, resulting in ten members representing five previously defined phylogenetic groups, i.e., TIP1-5. Although the gene amounts are equal to that present in two model plants Arabidopsis and rice, the group composition and/or evolution pattern were shown to be different. Except for CeTIP1;3 that has no counterpart in both Arabidopsis and rice, complex orthologous relationships of 1:1, 1:2, 1:3, 2:1, and 2:2 were observed. Expansion of the CeTIP subfamily was contributed by whole-genome duplication (WGD), transposed, and dispersed duplications. In contrast to the recent WGD-derivation of CeTIP3;1/-3;2, synteny analyses indicated that TIP4 and - 5 are old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. Expression analysis revealed that CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, when transiently overexpressed in tobacco leaves, CeTIP1;1 was shown to locate in the vacuolar membrane and function in homo/heteromultimer, whereas CeTIP2;1 is located in the cell membrane and only function in heteromultimer. Interestingly, CeTIP1;1 could mediate the tonoplast-localization of CeTIP2;1 via protein interaction, implying complex regulatory patterns. CONCLUSIONS: Our findings provide a global view of CeTIP genes, which provide valuable information for further functional analysis and genetic improvement through manipulating key members in tigernut.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

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.

Identification of two mutant JASON-RELATED genes associated with unreduced pollen production in potato.

KEY MESSAGE: Multiple QTLs control unreduced pollen production in potato. Two major-effect QTLs co-locate with mutant alleles of genes with homology to AtJAS, a known regulator of meiotic spindle orientation. In diploid potato the production of unreduced gametes with a diploid (2n) rather than a haploid (n) number of chromosomes has been widely reported. Besides their evolutionary important role in sexual polyploidisation, unreduced gametes also have a practical value for potato breeding as a bridge between diploid and tetraploid germplasm. Although early articles argued for a monogenic recessive inheritance, the genetic basis of unreduced pollen production in potato has remained elusive. Here, three diploid full-sib populations were genotyped with an amplicon sequencing approach and phenotyped for unreduced pollen production across two growing seasons. We identified two minor-effect and three major-effect QTLs regulating this trait. The two QTLs with the largest effect displayed a recessive inheritance and an additive interaction. Both QTLs co-localised with genes encoding for putative AtJAS homologs, a key regulator of meiosis II spindle orientation in Arabidopsis thaliana. The function of these candidate genes is consistent with the cytological phenotype of mis-oriented metaphase II plates observed in the parental clones. The alleles associated with elevated levels of unreduced pollen showed deleterious mutation events: an exonic transposon insert causing a premature stop, and an amino acid change within a highly conserved domain. Taken together, our findings shed light on the natural variation underlying unreduced pollen production in potato and will facilitate interploidy breeding by enabling marker-assisted selection for this trait.

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

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

A transient in planta editing assay identifies specific binding of the splicing regulator PTB as a prerequisite for cassette exon inclusion.

The dynamic interaction of RNA-binding proteins (RBPs) with their target RNAs contributes to the diversity of ribonucleoprotein (RNP) complexes that are involved in a myriad of biological processes. Identifying the RNP components at high resolution and defining their interactions are key to understanding their regulation and function. Expressing fusions between an RBP of interest and an RNA editing enzyme can result in nucleobase changes in target RNAs, representing a recent addition to experimental approaches for profiling RBP/RNA interactions. Here, we have used the MS2 protein/RNA interaction to test four RNA editing proteins for their suitability to detect target RNAs of RBPs in planta. We have established a transient test system for fast and simple quantification of editing events and identified the hyperactive version of the catalytic domain of an adenosine deaminase (hADARcd) as the most suitable editing enzyme. Examining fusions between homologs of polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana and hADARcd allowed determining target RNAs with high sensitivity and specificity. Moreover, almost complete editing of a splicing intermediate provided insight into the order of splicing reactions and PTB dependency of this particular splicing event. Addition of sequences for nuclear localisation of the fusion protein increased the editing efficiency, highlighting this approach's potential to identify RBP targets in a compartment-specific manner. Our studies have established the editing-based analysis of interactions between RBPs and their RNA targets in a fast and straightforward assay, offering a new system to study the intricate composition and functions of plant RNPs in vivo.

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts.

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.

Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export.

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.

Light-dependent expression and accumulation of miR408-encoded peptide, miPEP408, is regulated by HY5 in Arabidopsis.

Recent studies propose that primary transcripts of miRNAs (pri-miRNAs) contain small Open Reading Frames (ORFs) capable of encoding miRNA-encoded peptides (miPEPs). These miPEPs can function as transcriptional regulators for their corresponding pri-miRNAs, ultimately enhancing mature miRNA accumulation. Notably, pri-miR408 encodes the functional peptide miPEP408, regulating expression of miR408 and its target genes, providing plant tolerance to stresses. While miPEPs are crucial regulators, the factors governing them are have not been studied in detail. Here, we explored the light-dependent regulation of miPEP408 in Arabidopsis. Expression analysis during dark-light transitions revealed light-induced transcription and accumulation of the miPEP408. As the promoter of miR408 contains cis-acting elements responsible for binding to the bZIP-type transcription factor ELONGATED HYPOCOTYL5 (HY5), known for light-mediated regulation in plants, we studied its involvement in the regulation of miR408. Analysis of HY5 mutant (hy5-215), complemented line (HY5OX/hy5), and CONSTITUTIVE PHOTOMORPHOGENIC 1 mutant (cop1-4) plants supported HY5's positive regulation of miPEP408. Grafting and GUS assays further suggested the role of HY5 as a shoot-root mobile signal inducing light-dependent miPEP408 expression. This study underscores the regulatory impact of light on small peptides, exemplified by miPEP408, mediated by the key transcription factor HY5.

A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance.

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.

Ectopic expression of the Arabidopsis mutant L3 NB-LRR receptor gene in Nicotiana benthamiana cells leads to cell death.

Nucleotide-binding sites and leucine-rich repeat proteins (NLRs) act as critical intracellular immune receptors. Previous studies reported an Arabidopsis-resistant gene L3 (AT1G15890), which encoded a coiled-coil (CC) NLR that conferred cell death in bacteria; however, its function in planta remains unclear. This study describes a comprehensive structure-function analysis of L3 in Nicotiana benthamiana. The results of the transient assay showed that the L3 CC domain is sufficient for cell-death induction. The first 140 amino acid segment constituted the minimal function region that could cause cell death. The YFP-labeled L3 CC domain was localized to the plasma membrane, which was considered crucial for the function and self-interaction of the L3 CC domain. The results of point mutations analysis showed that L3 CC domain function is affected by mutations in some specific residues, and loss-of-function mutations in the CC domain affected the function of full-length L3. These study results offered considerable evidence to understand the activation mechanism of L3.

Defective pollen tube tip growth induces neo-polyploid infertility.

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

Endomembrane-biased dimerization of ABCG16 and ABCG25 transporters determines their substrate selectivity in ABA-regulated plant growth and stress responses.

ATP-binding cassette (ABC) transporters are integral membrane proteins that have evolved diverse functions fulfilled via the transport of various substrates. In Arabidopsis, the G subfamily of ABC proteins is particularly abundant and participates in multiple signaling pathways during plant development and stress responses. In this study, we revealed that two Arabidopsis ABCG transporters, ABCG16 and ABCG25, engage in ABA-mediated stress responses and early plant growth through endomembrane-specific dimerization-coupled transport of ABA and ABA-glucosyl ester (ABA-GE), respectively. We first revealed that ABCG16 contributes to osmotic stress tolerance via ABA signaling. More specifically, ABCG16 induces cellular ABA efflux in both yeast and plant cells. Using FRET analysis, we showed that ABCG16 forms obligatory homodimers for ABA export activity and that the plasma membrane-resident ABCG16 homodimers specifically respond to ABA, undergoing notable conformational changes. Furthermore, we demonstrated that ABCG16 heterodimerizes with ABCG25 at the endoplasmic reticulum (ER) membrane and facilitates the ER entry of ABA-GE in both Arabidopsis and tobacco cells. The specific responsiveness of the ABCG16-ABCG25 heterodimer to ABA-GE and the superior growth of their double mutant support an inhibitory role of these two ABCGs in early seedling establishment via regulation of ABA-GE translocation across the ER membrane. Our endomembrane-specific analysis of the FRET signals derived from the homo- or heterodimerized ABCG complexes allowed us to link endomembrane-biased dimerization to the translocation of distinct substrates by ABCG transporters, providing a prototypic framework for understanding the omnipotence of ABCG transporters in plant development and stress responses.