Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas.

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

The RALF signaling pathway regulates cell wall integrity during pollen tube growth in maize.

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Z. mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Z. mays LLG 1 and 2 (ZmLLG1/2), and Z. mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants.

Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Progressive chromatin silencing of ABA biosynthesis genes permits seed germination in Arabidopsis.

Seed germination represents a major developmental switch in plants that is vital to agriculture, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis thaliana requires a chromatin mechanism that progressively silences 9-CIS-EPOXYCAROTENOID DIOXYGENASE 6 (NCED6), which encodes a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocked germination and synergistically derepressed NCEDs and hundreds of genes. At NCED6, in part by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also an H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis revealed that RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination, when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate for and synergize with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via the RNA-binding protein RZ-1 and PRC2 acting in synergy, a process that is vital for seed germination.

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis.

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Egg cell-secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis.

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

The egg cell is preferentially fertilized in Arabidopsis double fertilization.

In flowering plants (angiosperms), fertilization of the egg cell by one sperm cell produces an embryo, whereas fusion of a second sperm cell with the central cell generates the endosperm. In most angiosperms like Arabidopsis, a pollen grain contains two isomorphic sperm cells required for this double fertilization process. A long-standing unsolved question is whether the two fertilization events have any preference. A tool to address this question is the usage of the cyclin-dependent kinase a1 (cdka;1) mutant pollen, which produces a single sperm-like cell (SLC). Here, we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC. Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell, precursor of the two sperm cells. Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2% of the ovules, single fertilization of the egg cell occurred. Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery, which fertilized the central cell, resulting in 20.7% double-fertilized ovules. This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell. Taken together, our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.

TANDEM ZINC-FINGER/PLUS3 Is a Key Component of Phytochrome A Signaling.

Phytochrome A (phyA) is the primary plant photoreceptor responsible for perceiving and mediating various responses to far-red (FR) light and is essential for survival in canopy shade. In this study, we identified two Arabidopsis thaliana mutants that grew longer hypocotyls in FR light. Genetic analyses showed that they were allelic and their FR phenotypes were caused by mutations in the gene named TANDEM ZINC-FINGER/PLUS3 (TZP), previously shown to encode a nuclear protein involved in blue light signaling and phyB-dependent regulation of photoperiodic flowering. We show that the expression of TZP is dramatically induced by light and that TZP proteins are differentially modified in different light conditions. Furthermore, we show that TZP interacts with both phyA and FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and regulates the abundance of phyA, FHY1, and ELONGATED HYPOCOTYL5 proteins in FR light. Moreover, our data indicate that TZP is required for the formation of a phosphorylated form of phyA in the nucleus in FR light. Together, our results identify TZP as a positive regulator of phyA signaling required for phosphorylation of the phyA photoreceptor, thus suggesting an important role of phosphorylated phyA in inducing the FR light response.

A Novel Imprinted Gene NUWA Controls Mitochondrial Function in Early Seed Development in Arabidopsis.

Imprinted genes display biased expression of paternal and maternal alleles and are only found in mammals and flowering plants. Compared to several hundred imprinted genes that are functionally characterized in mammals, very few imprinted genes were confirmed in plants and even fewer of them have been functionally investigated. Here, we report a new imprinted gene, NUWA, in plants. NUWA is an essential gene, because loss of its function resulted in reduced transmission through the female gametophyte and defective cell/nuclear proliferation in early Arabidopsis embryo and endosperm. NUWA is a maternally expressed imprinted gene, as only the maternal allele of NUWA is transcribed and translated from gametogenesis to the 16-cell globular embryo stage after fertilization, and the de novo transcription of the maternal allele of NUWA starts from the zygote stage. Different from other identified plant imprinted genes whose encoded proteins are mostly localized to the nucleus, the NUWA protein was localized to the mitochondria and was essential for mitochondria function. Our work uncovers a novel imprinted gene of a previously unidentified type, namely, a maternal-specific expressed nuclear gene with its encoded protein localizing to and controlling the function of the maternally inherited mitochondria. This reveals a unique mechanism of maternal control of the mitochondria and adds an extra layer of complexity to the regulation of nucleus-organelle coordination during early plant development.

Isocitrate lyase plays important roles in plant salt tolerance.

BACKGROUND: Isocitrate lyase (ICL) is a key enzyme in the glyoxylate cycle. In a previous study in rice, the expression of the ICL-encoding gene (OsICL) was highly induced by salt stress and its expression was enhanced in transgenic rice lines overexpressing OsCam1-1, a calmodulin (CaM)-encoding gene. CaM has been implicated in salt tolerance mechanisms in plants; however, the cellular mechanisms mediated by CaM are not clearly understood. In this study, the role of OsICL in plant salt tolerance mechanisms and the possible involvement of CaM were investigated using transgenic plants expressing OsICL or OsCam1-1. RESULTS: OsICL was highly expressed in senesced leaf and significantly induced by salt stress in three OsCam1-1 overexpressing transgenic rice lines as well as in wild type (WT). In WT young leaf, although OsICL expression was not affected by salt stress, all three transgenic lines exhibited highly induced expression levels. In Arabidopsis, salt stress had negative effects on germination and seedling growth of the AtICL knockout mutant (Aticl mutant). To examine the roles of OsICL we generated the following transgenic Arabidopsis lines: the Aticl mutant expressing OsICL driven by the native AtICL promoter, the Aticl mutant overexpressing OsICL driven by the 35SCaMV promoter, and WT overexpressing OsICL driven by the 35SCaMV promoter. Under salt stress, the germination rate and seedling fresh and dry weights of the OsICL-expressing lines were higher than those of the Aticl mutant, and the two lines with the icl mutant background were similar to the WT. The Fv/Fm and temperature of rosette leaves in the OsICL-expressing lines were less affected by salt stress than they were in the Aticl mutant. Finally, glucose and fructose contents of the Aticl mutant under salt stress were highest, whereas those of OsICL-expressing lines were similar to or lower than those of the WT. CONCLUSIONS: OsICL, a salt-responsive gene, was characterized in the transgenic Arabidopsis lines, revealing that OsICL expression could revert the salt sensitivity phenotypes of the Aticl knockout mutant. This work provides novel evidence that supports the role of ICL in plant salt tolerance through the glyoxylate cycle and the possible involvement of OsCam1-1 in regulating its transcription.

Pollen tube integrity regulation in flowering plants: insights from molecular assemblies on the pollen tube surface.

Contents Summary 687 I. Introduction 687 II. Pollen tube membrane-localized receptors coordinate cell integrity and sperm release 689 III. RALF peptides mediate autocrine and paracrine signaling 689 IV. ROS and ion channel signaling mediate intracellular response 690 V. Involvements from pollen tube cell wall components 690 VI. Concluding remarks 691 Acknowledgements 692 Author contributions 692 References 692 SUMMARY: Unlike in animals, sperm in flowering plants are immotile and they are embraced as passive cargoes by a pollen tube which embarks on a long journey in the pistil to deliver them to the female gametophyte for fertilization. How the pollen tube switches from a rapid polarized growth towards its target to an abrupt disintegration for sperm cell release inside the female gametophyte is puzzling. Recent studies have shown that members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family and their ligands, 5-kDa cysteine-rich peptide rapid alkalinization factors (RALFs), engage in an intricate balancing act involving autocrine and paracrine signaling to maintain pollen tube growth and induce timely tube rupture at the spatially confined pollen tube-female gametophyte interface. Here, we review recent progress related to pollen tube integrity control, mainly focusing on the molecular understanding of signaling as well as intracellular signaling nodes in Arabidopsis. Some missing links and future perspectives are also discussed.

RNA Binding Proteins RZ-1B and RZ-1C Play Critical Roles in Regulating Pre-mRNA Splicing and Gene Expression during Development in Arabidopsis.

Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here, we report the functions of two proteins containing RNA recognition motifs, RZ-1B and RZ-1C, in Arabidopsis thaliana. RZ-1B and RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C termini. RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA binding activity of RZ-1C with SR proteins through overexpression of the C terminus of RZ-1C conferred defective phenotypes similar to those observed in rz-1b rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of RZ-1B and RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that RZ-1C directly targeted FLOWERING LOCUS C (FLC), promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

AtVPS41-mediated endocytic pathway is essential for pollen tube-stigma interaction in Arabidopsis.

In flowering plants, extensive male-female interactions are required for successful fertilization in which various signaling cascades are involved. Prevacuolar compartments (PVC) and vacuoles are two types of subcellular compartments that terminate signal transduction by sequestrating signaling molecules in yeast and mammalian cells; however, the manner in which they might be involved in male-female interactions in plants is unknown. In this study, we identified Arabidopsis thaliana vacuolar protein sorting 41 (AtVPS41), encoded by a single-copy gene with sequence similarity to yeast Vps41p, as a new factor controlling pollen tube-stigma interaction. Loss of AtVPS41 function disrupted penetration of pollen tubes into the transmitting tissue and thus led to failed male transmission. In the pollen tubes, AtVPS41 protein is associated with PVCs and the tonoplast. We demonstrate that AtVPS41 is required for the late stage of the endocytic pathway (i.e., endomembrane trafficking from PVCs to vacuoles) because internalization of cell-surface molecules was normal in the vps41-deficient pollen tubes, whereas PVC-to-vacuole trafficking was impaired. We further show that the CHCR domain is required for subcellular localization and biological functioning of AtVPS41. These results indicate that the AtVPS41-mediated late stage of the endocytic pathway is essential for pollen tube-stigma interaction in Arabidopsis.

CFLAP1 and CFLAP2 Are Two bHLH Transcription Factors Participating in Synergistic Regulation of AtCFL1-Mediated Cuticle Development in Arabidopsis.

The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1) and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1.

Mechanism of DNA-Induced Phase Separation for Transcriptional Repressor VRN1.

Phase separation of proteins/nucleic acids to form non-membrane organelles is crucial in cellular gene-expression regulation. However, little is known about transcriptional regulator phase separation and the underlying molecular mechanism. Vernalization 1 (VRN1) encodes a crucial transcriptional repressor involved in plant vernalization that contains two B3 DNA-binding domains connected by an intrinsic disorder region (IDR) and nonspecifically binds DNA. We found that the Arabidopsis VRN1 protein undergoes liquid-liquid phase separation (LLPS) with DNA that is driven by multivalent protein-DNA interactions (LLPS), and that both B3 domains are required. The distribution of charged residues in the VRN1 IDR modulates the interaction strength between VRN1 and DNA, and changes in the charge pattern lead to interconversion between different states (precipitates, liquid droplets, and no phase separation). We further showed that VRN1 forms puncta in plant cell nuclei, suggesting that it may stabilize the vernalized state by repressing gene expression through LLPS.

GLABRA2 Directly Suppresses Basic Helix-Loop-Helix Transcription Factor Genes with Diverse Functions in Root Hair Development.

The Arabidopsis thaliana GLABRA2 (GL2) gene encodes a transcription factor involved in the cell differentiation of various epidermal tissues. During root hair pattern formation, GL2 suppresses root hair development in non-hair cells, acting as a node between the gene regulatory networks for cell fate determination and cell differentiation. Despite the importance of GL2 function, its molecular basis remains obscure because the GL2 target genes leading to the network for cell differentiation are unknown. We identified five basic helix-loop-helix (bHLH) transcription factor genes (ROOT HAIR DEFECTIVE6 [RHD6], RHD6-LIKE1 [RSL1], RSL2, Lj-RHL1-LIKE1 [LRL1], and LRL2) as GL2 direct targets using transcriptional and posttranslational induction systems. Chromatin immunoprecipitation analysis confirmed GL2 binding to upstream regions of these genes in planta. Reporter gene analyses showed that these genes are expressed in various stages of root hair development and are suppressed by GL2 in non-hair cells. GL2 promoter-driven GFP fusions of LRL1 and LRL2, but not those of the other bHLH proteins, conferred root hair development on non-hair cells. These results indicate that GL2 directly suppresses bHLH genes with diverse functions in root hair development.

The WRKY Transcription Factor WRKY71/EXB1 Controls Shoot Branching by Transcriptionally Regulating RAX Genes in Arabidopsis.

Plant shoot branching is pivotal for developmental plasticity and crop yield. The formation of branch meristems is regulated by several key transcription factors including REGULATOR OF AXILLARY MERISTEMS1 (RAX1), RAX2, and RAX3. However, the regulatory network of shoot branching is still largely unknown. Here, we report the identification of EXCESSIVE BRANCHES1 (EXB1), which affects axillary meristem (AM) initiation and bud activity. Overexpression of EXB1 in the gain-of-function mutant exb1-D leads to severe bushy and dwarf phenotypes, which result from excessive AM initiation and elevated bud activities. EXB1 encodes the WRKY transcription factor WRKY71, which has demonstrated transactivation activities. Disruption of WRKY71/EXB1 by chimeric repressor silencing technology leads to fewer branches, indicating that EXB1 plays important roles in the control of shoot branching. We demonstrate that EXB1 controls AM initiation by positively regulating the transcription of RAX1, RAX2, and RAX3. Disruption of the RAX genes partially rescues the branching phenotype caused by EXB1 overexpression. We further show that EXB1 also regulates auxin homeostasis in control of shoot branching. Our data demonstrate that EXB1 plays pivotal roles in shoot branching by regulating both transcription of RAX genes and auxin pathways.

ADP1 affects plant architecture by regulating local auxin biosynthesis.

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.