Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.

Leaf senescence is an essential physiological process related to grain yield potential and nutritional quality. Green leaf duration (GLD) after anthesis directly reflects the leaf senescence process and exhibits large genotypic differences in common wheat; however, the underlying gene regulatory mechanism is still lacking. Here, we identified TaNAM-A1 as the causal gene of the major loci qGLD-6A for GLD during grain filling by map-based cloning. Transgenic assays and TILLING mutant analyses demonstrated that TaNAM-A1 played a critical role in regulating leaf senescence, and also affected spike length and grain size. Furthermore, the functional divergences among the three haplotypes of TaNAM-A1 were systematically evaluated. Wheat varieties with TaNAM-A1d (containing two mutations in the coding DNA sequence of TaNAM-A1) exhibited a longer GLD and superior yield-related traits compared to those with the wild type TaNAM-A1a. All three haplotypes were functional in activating the expression of genes involved in macromolecule degradation and mineral nutrient remobilization, with TaNAM-A1a showing the strongest activity and TaNAM-A1d the weakest. TaNAM-A1 also modulated the expression of the senescence-related transcription factors TaNAC-S-7A and TaNAC016-3A. TaNAC016-3A enhanced the transcriptional activation ability of TaNAM-A1a by protein-protein interaction, thereby promoting the senescence process. Our study offers new insights into the fine-tuning of the leaf functional period and grain yield formation for wheat breeding under various geographical climatic conditions.

Most mechanistic details of chronologically ordered regulation of leaf senescence are unknown. Regulatory networks centered on AtWRKY53 are crucial for orchestrating and integrating various senescence-related signals. Notably, AtWRKY53 binds to its own promoter and represses transcription of AtWRKY53, but the biological significance and mechanism underlying this self-repression remain unclear. In this study, we identified the VQ motif-containing protein AtVQ25 as a cooperator of AtWRKY53. The expression level of AtVQ25 peaked at mature stage and was specifically repressed after the onset of leaf senescence. AtVQ25-overexpressing plants and atvq25 mutants displayed precocious and delayed leaf senescence, respectively. Importantly, we identified AtWRKY53 as an interacting partner of AtVQ25. We determined that interaction between AtVQ25 and AtWRKY53 prevented AtWRKY53 from binding to W-box elements on the AtWRKY53 promoter and thus counteracted the self-repression of AtWRKY53. In addition, our RNA-sequencing data revealed that the AtVQ25-AtWRKY53 module is related to the salicylic acid (SA) pathway. Precocious leaf senescence and SA-induced leaf senescence in AtVQ25-overexpressing lines were inhibited by an SA pathway mutant, atsid2, and NahG transgenic plants; AtVQ25-overexpressing/atwrky53 plants were also insensitive to SA-induced leaf senescence. Collectively, we demonstrated that AtVQ25 directly attenuates the self-repression of AtWRKY53 during the onset of leaf senescence, which is substantially helpful for understanding the timing of leaf senescence onset modulated by AtWRKY53.

Orchid plants develop protocorms upon germination and produce protocorm-like structures called protocorm-like bodies (PLBs) from protocorms and somatic cells via tissue culture. Protocorm-like bodies have broad technical application potential in the orchid industry and their regeneration is a distinct developmental process in the plant kingdom. However, little is known about this unparalleled developmental program. In this study, we identified a PLB-abundant gene, ethylene response factor (ERF), and a transcription factor named DoERF5, and determined its important role in PLB regeneration in Dendrobium orchid. Overexpression of DoERF5 in Dendrobium greatly enhanced the PLB regeneration from PLB and stem explants, and upregulated the expression of WOUND-INDUCED DEDIFFERENTIATION (DoWIND) homologs and SHOOT MERISTEMLESS (DoSTM), as well as the genes involved in cytokinin biosynthesis (DoIPT) and the cytokinin response factors (DoARRs). However, silencing DoERF5 reduced the regeneration rate of PLBs, and downregulated the expression of DoWIND homologs, DoSTM and DoARRs. We demonstrated that DoERF5 is directly bound to the DoSTM promoter and regulates its expression. In addition, overexpression of DoSTM in Dendrobium orchid resulted in favorable regeneration of PLBs. Our results clarify that DoERF5 regulates the regeneration of PLB by enhancing DoSTM expression. Our findings provide new insights into how DoERF5 mediates PLB regeneration and offers technical potential in improving clonal propagation, preservation, and the bioengineering of orchids.

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K⁺ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K⁺ homeostasis.

Flowering locus C (FLC) is a central transcriptional repressor that controls flowering time. However, how FLC is imported into the nucleus is unknown. Here, we report that Arabidopsis nucleoporins 62 (NUP62), NUP58, and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importin α-independent manner, via direct interaction. NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel. Importin β supersensitive to ABA and drought 2 (SAD2), a carrier protein, is critical for FLC nuclear import and flower transition, which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex. Proteomics, RNA-seq, and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences (NLSs), such as FLC. Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition, and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants.

Meiotic crossover (CO) formation between homologous chromosomes ensures their subsequent proper segregation and generates genetic diversity among offspring. In maize, however, the mechanisms that modulate CO formation remain poorly characterized. Here, we found that both maize BREAST CANCER SUSCEPTIBILITY PROTEIN 2 (BRCA2) and AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) act as positive factors of CO formation by controlling the assembly or/and stability of two conserved DNA recombinases RAD51 and DMC1 filaments. Our results revealed that ZmBRCA2 is not only involved in the repair of DNA double-stranded breaks (DSBs), but also regulates CO formation in a dosage-dependent manner. In addition, ZmFIGL1 interacts with RAD51 and DMC1, and Zmfigl1 mutants had a significantly reduced number of RAD51/DMC1 foci and COs. Further, simultaneous loss of ZmFIGL1 and ZmBRCA2 abolished RAD51/DMC1 foci and exacerbated meiotic defects compared with the single mutant Zmbrca2 or Zmfigl1. Together, our data demonstrate that ZmBRCA2 and ZmFIGL1 act coordinately to regulate the dynamics of RAD51/DMC1-dependent DSB repair to promote CO formation in maize. This conclusion is surprisingly different from the antagonistic roles of BRCA2 and FIGL1 in Arabidopsis, implying that, although key factors that control CO formation are evolutionarily conserved, specific characteristics have been adopted in diverse plant species.

Plants have adopted versatile scaffold proteins to facilitate the crosstalk between multiple signaling pathways. Leaf senescence is a well-programmed developmental stage that is coordinated by various external and internal signals. However, the functions of plant scaffold proteins in response to senescence signals are not well understood. Here, we report that the scaffold protein RACK1A (RECEPTOR FOR ACTIVATED C KINASE 1A) participates in leaf senescence mediated by ethylene signaling via the coordination of the EIN3-miR164-ORE1 transcriptional regulatory cascade. RACK1A is a novel positive regulator of ethylene-mediated leaf senescence. The rack1a mutant exhibits delayed leaf senescence, while transgenic lines overexpressing RACK1A display early leaf senescence. Moreover, RACK1A promotes EIN3 (ETHYLENE INSENSITIVE 3) protein accumulation, and directly interacts with EIN3 to enhance its DNA-binding activity. Together, they then associate with the miR164 promoter to inhibit its transcription, leading to the release of the inhibition on downstream ORE1 (ORESARA 1) transcription and the promotion of leaf senescence. This study reveals a mechanistic framework by which RACK1A promotes leaf senescence via the EIN3-miR164-ORE1 transcriptional cascade, and provides a paradigm for how scaffold proteins finely tune phytohormone signaling to control plant development.

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.

Anthocyanins are important natural plant pigments and play diverse roles in plant growth and adaptation. Anthocyanins function as screens to protect photosynthetic tissues from photoinhibition. However, the regulatory mechanisms underlying the biosynthesis and spatial accumulation pattern of anthocyanins remain some unresolved issues. Here, we demonstrate that the GARP-type transcription factor GOLDEN2-LIKE 1 (GLK1) functions as a positive factor in anthocyanin accumulation. GLK1 enhances the transcriptional activation activities of MYB75, MYB90, and MYB113 via direct protein- protein interactions to increase the expression of anthocyanin-specific biosynthetic genes. Anthocyanins accumulate in an acropetal manner in Arabidopsis. We also found that the expression pattern of GLK1 overall mimicked the accumulation pattern of anthocyanin from the base of the main stem to the shoot apex. Based on these findings, we established a working model for the role of GLK1 in anthocyanin accumulation and propose that GLK1 mediates the spatial distribution pattern of anthocyanins by affecting the transcriptional activation activities of MYB75, MYB90, and MYB113.

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal- anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co- translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.

Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In this study, we showed that the gene encoding an APETALA 2/ethylene-responsive element binding factor (AP2/ERF) transcription factor, PagERF81, from poplar 84?K (Populus alba × P. glandulosa) is highly expressed in expanding secondary xylem cells. Two independent homozygous Pagerf81 mutant lines created by gene editing, produced significantly more but smaller vessel cells and longer fiber cells with more lignin in cell walls, while PagERF81 overexpression lines had less lignin, compared to non-transgenic controls. Transcriptome and reverse transcription quantitative PCR data revealed that multiple lignin biosynthesis genes including Cinnamoyl CoA reductase 1 (PagCCR1), Cinnamyl alcohol dehydrogenase 6 (PagCAD6), and 4-Coumarate-CoA ligase-like 9 (Pag4CLL9) were up-regulated in Pagerf81 mutants, but down-regulated in PagERF81 overexpression lines. In addition, a transient transactivation assay revealed that PagERF81 repressed the transcription of these three genes. Furthermore, yeast one hybrid and electrophoretic mobility shift assays showed that PagERF81 directly bound to a GCC sequence in the PagCCR1 promoter. No known vessel or fiber cell differentiation related genes were differentially expressed, so the smaller vessel cells and longer fiber cells observed in the Pagerf81 lines might be caused by abnormal lignin deposition in the secondary cell walls. This study provides insight into the regulation of lignin biosynthesis, and a molecular tool to engineer wood with high lignin content, which would contribute to the lignin-related chemical industry and carbon sequestration.

Leaf senescence is the final stage of leaf development and appropriate onset and progression of leaf senescence are critical for reproductive success and fitness. Although great progress has been made in identifying key genes regulating leaf senescence and elucidating the underlining mechanisms in the model plant Arabidopsis, there is still a gap to understanding the complex regulatory network. In this study, we discovered that Arabidopsis ANAC087 transcription factor (TF) positively modulated leaf senescence. Expression of ANAC087 was induced in senescing leaves and the encoded protein acted as a transcriptional activator. Both constitutive and inducible overexpression lines of ANAC087 showed earlier senescence than control plants, whereas T‐DNA insertion mutation and dominant repression of the ANAC087 delayed senescence rate. A quantitative reverse transcription‐polymerase chain reaction (qRT‐ PCR) profiling showed that the expression of an array of senescence‐associated genes was upregulated in inducible ANAC087 overexpression plants including BFN1, NYE1, CEP1, RbohD, SAG13, SAG15, and VPEs, which are involved in programmed cell death (PCD), chlorophyll degradation and reactive oxygen species (ROS) accumulation. In addition, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation‐quantitative polymerase chain reaction (ChIP‐qPCR) assays demonstrated that ANAC087 directly bound to the canonical NAC recognition sequence (NACRS) motif in promoters of its target genes. Moreover, mutation of two representative target genes, BFN1 or NYE1 alleviated the senescence rate of ANAC087‐overexpression plants, suggesting their genetic regulatory relationship. Taken together, this study indicates that ANAC087 serves as an important regulator linking PCD, ROS, and chlorophyll degradation to leaf senescence.

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule dual-localized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell (PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation width, decreased lobe length, and similar lobe number of PCs, whereas IQD21 overexpression had a different effect on cotyledon PC shape. Weak overexpression led to increased lobe number, decreased indentation width, and similar lobe length, while moderate or great overexpression resulted in decreased lobe number, indentation width, and lobe length of PCs. Live-cell observations revealed that IQD21 accumulation at indentation regions correlates with lobe initiation and outgrowth during PC development. Cell biological and genetic approaches revealed that IQD21 promotes transfacial microtubules anchoring to the plasma membrane via its polybasic sites and bundling at the indentation regions in both periclinal and anticlinal walls. IQD21 controls cortical microtubule organization mainly through promoting Katanin 1-mediated microtubule severing during PC interdigitation. These findings provide the genetic evidence that transfacial microtubule arrays play a determinant role in lobe formation, and the insight into the molecular mechanism of IQD21 in transfacial microtubule organization at indentations and puzzle-shaped PC development.

Auxin is a crucial phytohormone that has various effects on the regulators of plant growth and development. Auxin signal transduction is mainly controlled by two gene families: auxin response factor (ARF) and auxin/indole-3-acetic acid (Aux/IAA). ARFs are plant-specific transcription factors that bind directly to auxin response elements in the promoters of auxin-responsive genes. ARF proteins contain three conserved regions: a conserved N-terminal B3 DNA-binding domain, a variable intermediate middle region domain that functions in activation or repression, and a C-terminal domain including the Phox and Bem1p region for dimerization, similar to the III and IV elements of Aux/IAA, which facilitate protein–protein interaction through homodimerization of ARF proteins or heterodimerization of ARF and Aux/IAA proteins. In the two decades following the identification of the first ARF, 23 ARF members have been identified and characterized in Arabidopsis. Using whole-genome sequencing, 22, 25, 23, 25, and 36 ARF genes have been identified in tomato, rice, wheat, sorghum, and maize, respectively, in addition to which the related biofunctions of some ARFs have been reported. ARFs play crucial roles in regulating the growth and development of roots, leaves, flowers, fruits, seeds, responses to biotic and abiotic stresses, and phytohormone signal crosstalk. In this review, we summarize the research progress on the structures and functions of ARFs in Arabidopsis, tomato, and cereal crops, to provide clues for future basic research on phytohormone signaling and the molecular design breeding of crops.

Plant cells contain only small amounts of mitochondrial DNA (mtDNA), with the genomic information shared among multiple mitochondria. The biological relevance and molecular mechanism underlying this hallmark of plant cells has been unclear. Here, we report that Arabidopsis thaliana plants exhibited significantly reduced growth and mitochondrial dysfunction when the mtDNA copy number was increased to the degree that each mitochondrion possessed DNA. The amounts of mitochondrion-encoded transcripts increased several fold in the presence of elevated mtDNA levels. However, the efficiency of RNA editing decreased with this excess of mitochondrion-encoded transcripts, resulting in impaired assembly of mitochondrial complexes containing mtDNA-encoded subunits, such as respiratory complexes I and IV. These observations indicate the occurrence of nuclear–mitochondrial incompatibility in the cells with increased amounts of mtDNA and provide an initial answer to the fundamental question of why plant cells have much lower mtDNA levels than animal cells. We propose that keeping mtDNA levels low moderates nuclear–mitochondrial incompatibility and that this may be a crucial factor driving plant cells to restrict the copy numbers of mtDNA.

Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins (AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine (COR), enhanced maize (Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5 (ZmPIP2;5). In vivo and in vitro experiments indicated that COR also directly acts on ZmPIP2;5 to improve water uptake in maize and Xenopus oocytes. The leaf water potential and hydraulic conductivity of roots growing under hyperosmotic conditions were higher in ZmPIP2;5-overexpression lines and lower in the zmpip2;5 knockout mutant, compared to wild-type plants. Based on a comparison between ZmPIP2;5 and other PIP2s, we predicted that COR may bind to the functional site in loop E of ZmPIP2;5. We confirmed this prediction by surface plasmon resonance technology and a microscale thermophoresis assay, and showed that deleting the binding motif greatly reduced COR binding. We identified the N241 residue as the COR-specific binding site, which may activate the channel of the AQP tetramer and increase water transport activity, which may facilitate water uptake under hyperosmotic stress.

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.

Camptothecin (CPT) is an anticancer pentacyclic quinoline alkaloid widely used to treat cancer patients worldwide. However, the biosynthetic pathway and transcriptional regulation of camptothecin are largely unknown. Ophiorrhiza pumila, the herbaceous plant from the Rubiaceae family, has emerged as a model plant for studying camptothecin biosynthesis and regulation. In this study, a high-quality reference genome of O. pumila with estimated size of ~456.90?Mb was reported, and the accumulation level of camptothecin in roots was higher than that in stems and leaves. Based on its spatial distribution in the plant, we examined gene functions and expression by combining genomics with transcriptomic analysis. Two loganic acid O-methyltransferase (OpLAMTs) were identified in strictosidine-producing plant O. pumila, and enzyme catalysis assays showed that OpLAMT1 and not OpLAMT2 could convert loganic acid into loganin. Further knock-out of OpLAMT1 expression led to the elimination of loganin and camptothecin accumulation in O. pumila hairy roots. Four key residues were identified in OpLAMT1 protein crucial for the catalytic activity of loganic acid to loganin. By co-expression network, we identified a NAC transcription factor, OpNAC1, as a candidate gene for regulating camptothecin biosynthesis. Transgenic hairy roots and biochemical assays demonstrated that OpNAC1 suppressed OpLAMT1 expression. Here, we reported on two camptothecin metabolic engineering strategies paving the road for industrial-scale production of camptothecin in CPT-producing plants.

Repression of embryonic traits during the seed-to-seedling phase transition requires the inactivation of master transcription factors associated with embryogenesis. How the timing of such inactivation is controlled is unclear. Here, we report on a novel transcriptional co-repressor, Arabidopsis thaliana SDR4L, that forms a feedback inhibition loop with the master transcription factors LEC1 and ABI3 to repress embryonic traits post-imbibition. LEC1 and ABI3 regulate their own expression by inducing AtSDR4L during mid to late embryogenesis. AtSDR4L binds to sites upstream of LEC1 and ABI4, and these transcripts are upregulated in Atsdr4l seedlings. Atsdr4l seedlings phenocopy a LEC1 overexpressor. The embryonic traits of Atsdr4l can be partially rescued by impairing LEC1 or ABI3. The penetrance and expressivity of the Atsdr4l phenotypes depend on both developmental and external cues, demonstrating the importance of AtSDR4L in seedling establishment under suboptimal conditions.

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.

Faithful DNA replication is one of the most essential processes in almost all living organisms. However, the proteins responsible for organellar DNA replication are still largely unknown in plants. Here, we show that the two mitochondrion-targeted single-stranded DNA-binding (SSB) proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA (mtDNA) maintenance, as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation. The mtDNA levels in mutants lacking SSB1 and/or SSB2 function were two- to four-fold higher than in the wild-type (WT), revealing a negative role for SSB1/2 in regulating mtDNA replication. Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA (POLIA) or POLIB in mtDNA replication, as mutation in either gene restored the high mtDNA copy number of the ssb1-1 mutant back to WT levels. In addition, SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mtDNA homologous recombination (HR). Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNA-BINDING PROTEIN1 (OSB1) during mtDNA replication, while SSB1 may act downstream of OSB1 and MUTS HOMOLOG1 for mtDNA HR. Overall, our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mtDNA stability via affecting DNA replication and DNA HR.

The ginsenoside Rg₃ found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg₃ in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg₃ from Rh₂. Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg₃. Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg₃ accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg₃ accumulation. Finally, we produced ginsenoside Rg₃ at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg₃ commercially for pharmaceutical use.

Endoreduplication is prevalent during plant growth and development, and is often correlated with large cell and organ size. Despite its prevalence, the transcriptional regulatory mechanisms underlying the transition from mitotic cell division to endoreduplication remain elusive. Here, we characterize ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR 4 (ERF4) as a positive regulator of endoreduplication through its function as a transcriptional repressor. ERF4 was specifically expressed in mature tissues in which the cells were undergoing expansion, but was rarely expressed in young organs. Plants overexpressing ERF4 exhibited much larger cells and organs, while plants that lacked functional ERF4 displayed smaller organs than the wild-type. ERF4 was further shown to regulate cell size by controlling the endopolyploidy level in the nuclei. Moreover, ERF4 physically associates with the class I TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) protein TCP15, a transcription factor that inhibits endoreduplication by activating the expression of a key cell-cycle gene, CYCLIN A2;3 (CYCA2;3). A molecular and genetic analysis revealed that ERF4 promotes endoreduplication by directly suppressing the expression of CYCA2;3. Together, this study demonstrates that ERF4 and TCP15 function as a module to antagonistically regulate each other's activity in regulating downstream genes, thereby controlling the switch from the mitotic cell cycle to endoreduplication during leaf development. These findings expand our understanding of how the control of the cell cycle is fine-tuned by an ERF4–TCP15 transcriptional complex.

The organization of the microtubule cytoskeleton is critical for cell and organ morphogenesis. The evolutionarily conserved microtubule-severing enzyme KATANIN plays critical roles in microtubule organization in the plant and animal kingdoms. We previously used conical cell of Arabidopsis thaliana petals as a model system to investigate cortical microtubule organization and cell morphogenesis and determined that KATANIN promotes the formation of circumferential cortical microtubule arrays in conical cells. Here, we demonstrate that the conserved protein phosphatase PP2A interacts with and dephosphorylates KATANIN to promote the formation of circumferential cortical microtubule arrays in conical cells. KATANIN undergoes cycles of phosphorylation and dephosphorylation. Using co-immunoprecipitation coupled with mass spectrometry, we identified PP2A subunits as KATANIN-interacting proteins. Further biochemical studies showed that PP2A interacts with and dephosphorylates KATANIN to stabilize its cellular abundance. Similar to the katanin mutant, mutants for genes encoding PP2A subunits showed disordered cortical microtubule arrays and defective conical cell shape. Taken together, these findings identify PP2A as a regulator of conical cell shape and suggest that PP2A mediates KATANIN phospho-regulation during plant cell morphogenesis.