Engineered ATG8-binding motif-based selective autophagy to degrade proteins and organelles in planta.

Protein-targeting technologies represent essential approaches in biological research. Protein knockdown tools developed recently in mammalian cells by exploiting natural degradation mechanisms allow for precise determination of protein function and discovery of degrader-type drugs. However, no method to directly target endogenous proteins for degradation is currently available in plants. Here, we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescence-tagged proteins, including cytosolic mCherry, the nucleus-localized bZIP transcription factor TGA5, and the plasma membrane-anchored brassinosteroid receptor BRI1, as well as fluorescence-coated peroxisomes, using a tobacco-based transient expression system. Stable expression of AIM-based autophagy receptors in Arabidopsis further confirms the feasibility of this approach in selective autophagy of endogenous proteins. With its wide substrate scope and its specificity, our concept of engineered AIM-based selective autophagy could provide a convenient and robust research tool for manipulating endogenous proteins in plants and may open an avenue toward degradation of cytoplasmic components other than proteins in plant research.

The RAC/ROP GTPase activator OsRopGEF10 functions in crown root development by regulating cytokinin signaling in rice.

RAC/Rho of plant (ROP) GTPases are major molecular switches that control diverse signaling cascades for plant growth, development, and defense. Here, we discovered a signaling node that connects RAC/ROPs to cytokinins. Rice (Oryza sativa) plants develop a fibrous root system mainly composed of crown roots. Cytokinin signaling via a phosphorelay system is critical for crown root development. We show that OsRopGEF10, which activates RAC/ROPs, acts upstream of the cytoplasmic-nuclear shuttling phosphotransfer proteins AHPs of the cytokinin signaling pathway to promote crown root development. Mutations of OsRopGEF10 induced hypersensitivity to cytokinin, whereas overexpressing this gene reduced the cytokinin response. Loss of OsRopGEF10 function reduced the expression of the response regulator gene OsRR6, a repressor of cytokinin signaling, and impaired crown root development. Mutations in OsAHP1/2 led to increased crown root production and rescued the crown root defect of Osropgef10. Furthermore, auxin activates the ROP GTPase OsRAC3, which attenuates cytokinin signaling for crown root initiation. Molecular interactions between OsRopGEF10, OsRAC3, and OsAHP1/2 implicate a mechanism whereby OsRopGEF10-activated OsRAC3 recruits OsAHP1/2 to the cortical cytoplasm, sequestering them from their phosphorelay function in the nucleus. Together, our findings uncover the OsRopGEF10-OsRAC3-OsAHP1/2 signaling module, establish a link between RAC/ROPs and cytokinin, and reveal molecular crosstalk between auxin and cytokinin during crown root development.

eIF4E1 Regulates Arabidopsis Embryo Development and Root Growth by Interacting With RopGEF7.

Eukaryotic translation initiation factor 4E1 (eIF4E1) is required for the initiation of protein synthesis. The biological function of eIF4E1 in plant-potyvirus interactions has been extensively studied. However, the role of eIF4E1 in Arabidopsis development remains unclear. In this study, we show that eIF4E1 is highly expressed in the embryo and root apical meristem. In addition, eIF4E1 expression is induced by auxin. eIF4E1 mutants show embryonic cell division defects and short primary roots, a result of reduced cell divisions. Furthermore, our results show that mutation in eIF4E1 severely reduces the accumulation of PIN-FORMED (PIN) proteins and decreases auxin-responsive gene expression at the root tip. Yeast two-hybrid assays identified that eIF4E1 interacts with an RAC/ROP GTPase activator, RopGEF7, which has been previously reported to be involved in the maintenance of the root apical meristem. The interaction between eIF4E1 and RopGEF7 is confirmed by protein pull-down and bimolecular fluorescent complementation assays in plant cells. Taken together, our results demonstrated that eIF4E1 is important for auxin-regulated embryo development and root growth. The eIF4E1-RopGEF7 interaction suggests that eIF4E1 may act through ROP signaling to regulate auxin transport, thus regulating auxin-dependent patterning.

Interactions Among Multiple Quantitative Trait Loci Underlie Rhizome Development of Perennial Rice.

Perennial crops have some advantages over annuals in soil erosion prevention, lower labor and water requirements, carbon sequestration, and maintenance of thriving soil ecosystems. Rhizome, a kind of root-like underground stem, is a critical component of perenniality, which allows many grass species to survive through harsh environment. Identification of rhizome-regulating genes will contribute to the development of perennial crops. There have been no reports on the cloning of such genes until now, which bring urgency for identification of genes controlling rhizomatousness. Using rhizomatous Oryza longistaminata and rhizome-free cultivated rice as male and female parents, respectively, genetic populations were developed to identify genes regulating rhizome. Both entire population genotyping and selective genotyping mapping methods were adopted to detect rhizome-regulating quantitative trait loci (QTL) in 4 years. Results showed that multiple genes regulated development of rhizomes, with over 10 loci related to rhizome growth. At last, five major-effect loci were identified including qRED1.2, qRED3.1, qRED3.3, qRED4.1, and qRED4.2. It has been found that the individual plant with well-developed rhizomes carried at least three major-effect loci and a certain number of minor-effect loci. Both major-effect and minor-effect loci worked together to control rhizome growth, while no one could work alone. These results will provide new understanding of genetic regulation on rhizome growth and reference to the subsequent gene isolation in rice. And the related research methods and results in this study will contribute to the research on rhizome of other species.

Phosphoethanolamine N-methyltransferase 1 contributes to maintenance of root apical meristem by affecting ROS and auxin-regulated cell differentiation in Arabidopsis.

The continuous growth of roots requires the balance between cell division and differentiation. Reactive oxygen species (ROS) and auxin are important regulators of root development by affecting cell division and differentiation. The mechanism controlling the coordination of cell division and differentiation is not well understood. Using a forward genetic screen, we isolated a mutant, defective primary root 2 (dpr2), defective in root apical meristem (RAM) maintenance. The DPR2 gene encodes phosphoethanolamine N-methyltransferase 1 (PEAMT1) that catalyzes phosphocholine biosynthesis in Arabidopsis. We characterized the primary root phenotypes of dpr2 using various marker lines, using histochemical and pharmacological analysis to probe early root development. Loss-of-function of DPR2/PEAMT1 resulted in RAM consumption by affecting root stem cell niche, division zone, elongation and differentiation zone (EDZ). PIN-FORMED (PIN) protein abundance, PIN2 polar distribution and general endocytosis were impaired in the root tip of dpr2. Excess hydrogen peroxide and auxin accumulate in the EDZ of dpr2, leading to RAM consumption by accelerating cell differentiation. Suppression of ROS over-accumulation or inhibition of auxin signalling partially prevent RAM differentiation in dpr2 after choline starvation. Taken together, we conclude that the EDZ of the root tip is most sensitive to choline shortage, leading to RAM consumption through an ROS-auxin regulation module.

Cystathionine beta-lyase is crucial for embryo patterning and the maintenance of root stem cell niche in Arabidopsis.

The growth and development of roots in plants depends on the specification and maintenance of the root apical meristem. Here, we report the identification of CBL, a gene required for embryo and root development in Arabidopsis, and encodes cystathionine beta-lyase (CBL), which catalyzes the penultimate step in methionine (Met) biosynthesis, and which also led to the discovery of a previous unknown, but crucial, metabolic contribution by the Met biosynthesis pathway. CBL is expressed in embryos and shows quiescent center (QC)-enriched expression pattern in the root. cbl mutant has impaired embryo patterning, defective root stem cell niche, stunted root growth, and reduces accumulation of the root master regulators PLETHORA1 (PLT1) and PLT2. Furthermore, mutation in CBL severely decreases abundance of several PIN-FORMED (PIN) proteins and impairs auxin-responsive gene expression in the root tip. cbl seedlings also exhibit global reduction in histone H3 Lys-4 trimethylation (H3K4me3) and DNA methylation. Importantly, mutation in CBL reduces the abundance of H3K4me3 modification in PLT1/2 genes and downregulates their expression. Overexpression of PLT2 partially rescues cbl root meristem defect, suggesting that CBL acts in part through PLT1/2. Moreover, exogenous supplementation of Met also restores the impaired QC activity and the root growth defects of cbl. Taken together, our results highlight the unique role of CBL to maintain the root stem cell niche by cooperative actions between Met biosynthesis and epigenetic modification of key developmental regulators.

FERONIA regulates auxin-mediated lateral root development and primary root gravitropism.

The Arabidopsis FERONIA (FER) receptor kinase is a key hub of cell signaling networks mediating various hormone, stress, and immune responses. Previous studies have shown that FER functions correlate with auxin responses, but the underlying molecular mechanism is unknown. Here, we demonstrate that the primary root of the fer-4 mutant displays increased lateral root branching and a delayed gravitropic response, which are associated with polar auxin transport (PAT). Our data suggest that aberrant PIN2 polarity is responsible for the delayed gravitropic response in fer-4. Furthermore, the diminished F-actin cytoskeleton in fer-4 implies that FER modulates F-actin-mediated PIN2 polar localization. Our findings provide new insights into the function of FER in PAT.

RIBOSE PHOSPHATE ISOMERSASE 1 Influences Root Development by Acting on Cell Wall Biosynthesis, Actin Organization, and Auxin Transport in Arabidopsis.

Cell wall biosynthesis plays essential roles in cell division and expansion and thus is fundamental to plant growth and development. In this work, we show that an Arabidopsis mutant dpr3, isolated by a forward genetic screen, displays embryo defects and short, swelling primary root with the failure of maintenance of root apical meristem reminiscent to several cell wall-deficient mutants. Map-based cloning identified dpr3 is a mutant allele of RIBOSE PHOSPHATE ISOMERSASE 1 (RPI1), an enzyme involved in cellulose synthesis. Cellulose content in the mutant was dramatically decreased. Moreover, dpr3 (rpi1 from hereon) caused aberrant auxin distribution, as well as defective accumulation of root master regulators PLETHORA (PLT1 and PLT2) and misexpression of auxin response factor 5 (MONOPTEROS, MP). The abnormal auxin distribution is likely due to the reduced accumulation of auxin efflux transporters PIN-FORMED (PIN1 and PIN3). Surprisingly, we found that the orientation of actin microfilaments was severely altered in rpi1 root cells, whereas the cortical microtubules stay normal. Our study provides evidence that the defects in cellulose synthesis in rpi1 affect polar auxin transport possibly connected with altered F-actin organization, which is critically important for vesicle trafficking, thus exerting effects on auxin distribution, signaling, and auxin-mediated plant development.

Guanine Nucleotide Exchange Factor 7B (RopGEF7B) is involved in floral organ development in Oryza sativa.

BACKGROUND: RAC/ROP GTPase are versatile signaling molecules controlling diverse biological processes including cell polarity establishment, cell growth, morphogenesis, hormone responses and many other cellular processes in plants. The activities of ROPs are positively regulated by guanine nucleotide exchange factors (GEFs). Evidence suggests that RopGEFs regulate polar auxin transport and polar growth in pollen tube in Arabidopsis thaliana. However, the biological functions of rice RopGEFs during plant development remain largely unknown. RESULTS: We investigated a member of the OsRopGEF family, namely OsRopGEF7B. OsRopGEF7Bpro:GUS analysis indicates that OsRopGEF7B is expressed in various tissues, especially in the floral meristem and floral organ primordia. Knock-out and -down of OsRopGEF7B by T-DNA insertion and RNA interference, respectively, predominantly caused an increase in the number of floral organs in the inner whorls (stamen and ovary), as well as abnormal paleae/lemmas and ectopic growth of lodicules, resulting in decline of rice seed setting. Bimolecular fluorescence complement (BiFC) assays as well as yeast two-hybrid assays indicate that OsRopGEF7B interacts with OsRACs. CONCLUSIONS: OsRopGEF7B plays roles in floral organ development in rice, affecting rice seed setting rate. Manipulation of OsRopGEF7B has potential for application in genetically modified crops.

RopGEF1 Plays a Critical Role in Polar Auxin Transport in Early Development.

Polar auxin transport, facilitated by the combined activities of auxin influx and efflux carriers to maintain asymmetric auxin distribution, is essential for plant growth and development. Here, we show that Arabidopsis (Arabidopsis thaliana) RopGEF1, a guanine nucleotide exchange factor and activator of Rho GTPases of plants (ROPs), is critically involved in polar distribution of auxin influx carrier AUX1 and differential accumulation of efflux carriers PIN7 and PIN2 and is important for embryo and early seedling development when RopGEF1 is prevalently expressed. Knockdown or knockout of RopGEF1 induces embryo defects, cotyledon vein breaks, and delayed root gravity responses. Altered expression from the auxin response reporter DR5rev:GFP in the root pole of RopGEF1-deficient embryos and loss of asymmetric distribution of DR5rev:GFP in their gravistimulated root tips suggest that auxin distribution is affected in ropgef1 mutants. This is reflected by the polarity of AUX1 being altered in ropgef1 embryos and roots, shifting from the normal apical membrane location to a basal location in embryo central vascular and root protophloem cells and also reduced PIN7 accumulation at embryos and altered PIN2 distribution in gravistimulated roots of mutant seedlings. In establishing that RopGEF1 is critical for AUX1 localization and PIN differential accumulation, our results reveal a role for RopGEF1 in cell polarity and polar auxin transport whereby it imapcts auxin-mediated plant growth and development.

ROP3 GTPase contributes to polar auxin transport and auxin responses and is important for embryogenesis and seedling growth in Arabidopsis.

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.

RopGEF7 regulates PLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis.

The root stem cell niche defines the area that specifies and maintains the stem cells and is essential for the maintenance of root growth. Here, we characterize and examine the functional role of a quiescent center (QC)-expressed RAC/ROP GTPase activator, RopGEF7, in Arabidopsis thaliana. We show that RopGEF7 interacts with At RAC1 and overexpression of a C-terminally truncated constitutively active RopGEF7 (RopGEF7ΔC) activates RAC/ROP GTPases. Knockdown of RopGEF7 by RNA interference causes defects in embryo patterning and maintenance of the QC and leads to postembryonic loss of root stem cell population. Gene expression studies indicate that RopGEF7 is required for root meristem maintenance as it regulates the expression of PLETHORA1 (PLT1) and PLT2, which are key transcription factors that mediate the patterning of the root stem cell niche. Genetic analyses show that RopGEF7 interacts with PLT genes to regulate QC maintenance. Moreover, RopGEF7 is induced transcriptionally by auxin while its function is required for the expression of the auxin efflux protein PIN1 and maintenance of normal auxin maxima in embryos and seedling roots. These results suggest that RopGEF7 may integrate auxin-derived positional information in a feed-forward mechanism, regulating PLT transcription factors and thereby controlling the maintenance of root stem cell niches.

RAC GTPases in tobacco and Arabidopsis mediate auxin-induced formation of proteolytically active nuclear protein bodies that contain AUX/IAA proteins.

Auxin signaling relies on ubiquitin ligase SCF(TIR1)-mediated 26S proteasome-dependent proteolysis of a large family of short-lived transcription regulators, auxin/indole acetic acid (Aux/IAA), resulting in the derepression of auxin-responsive genes. We have shown previously that a subset of Rac GTPases is activated by auxin, and they in turn stimulate auxin-responsive gene expression. We show here that increasing Rac signaling activity promotes Aux/IAA degradation, whereas downregulating that activity results in the reduction of auxin-accelerated Aux/IAA proteolysis. Observations reported here reveal a novel function for these Rac GTPases as regulators for ubiquitin/26S proteasome-mediated proteolysis and further consolidate their role in auxin signaling. Moreover, our study reveals a cellular process whereby auxin induces and Rac GTPases mediate the recruitment of nucleoplasmic Aux/IAAs into proteolytically active nuclear protein bodies, into which components of the SCF(TIR1), COP9 signalosome, and 26S proteasome are also recruited.

Regulation of pollen tube growth by Rac-like GTPases.

Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.

Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression.

The auxin indole-3-acetic acid is a key plant hormone essential for a broad range of growth and developmental processes. Here, we show that auxin activates Rac-like GTPases (referred to as Rac/Rop GTPases), and they in turn stimulate auxin-responsive gene expression. In particular, we show that overexpressing a wild-type tobacco Rac/Rop GTPase, NtRac1, and its constitutively active mutant form activates auxin-responsive gene expression. On the other hand, overexpressing dominant-negative NtRac1 and Rac-negative regulators, or reducing the endogenous NtRac1 level, suppresses auxin-induced gene expression. Furthermore, overexpression of NtRac1 activity or suppression of its expression in transgenic seedlings induces phenotypes that are similar to auxin-related defects. Together, our results show that a subset of plant Rac/Rop GTPases functions in mediating the auxin signal to downstream responsive genes.