Improving Seed Shattering Resistance in Wild O. alta Rice with Mesoporous Silica Nanoparticle Delivery Systems.

To address food security challenges and climate change, the polyploid wild rice Oryza alta has been explored as a potential crop, although it suffers from seed shattering. We employed mesoporous silica nanoparticles (MSNs) to deliver small interfering RNAs (siRNAs) for targeted gene silencing. Foliar spraying of MSN-siRNA complexes effectively delivered siRNA, resulting in up to 70% gene silencing of the PDS gene and 75% silencing of the transgenic Ruby gene. Additionally, MSN-siRNAs were infiltrated into the panicles of O. alta to target four seed shattering major genes every other day for 2 weeks until heading outdoors. This method silenced all four shattering genes ranging from 10.7% to 49.4% and significantly reduced the formation of the abscission layer between rice grains and pedicels, which enhanced pedicel tensile strength. Our MSN-siRNA system provides a flexible, nonpermanent approach to modifying crop traits, offering a promising tool for sustainable agricultural practices.

LAZY4 acts additively with the starch-statolith-dependent gravity-sensing pathway to regulate shoot gravitropism and tiller angle in rice.

Rice tiller angle is a key agronomic trait that has significant effects on the establishment of a high-yield rice population. However, the molecular mechanism underlying the control of rice tiller angle remains to be clarified. Here, we characterized the novel tiller-angle gene LAZY4 (LA4) in rice through map-based cloning. LA4 encodes a C3H2C3-type RING zinc-finger E3 ligase localized in the nucleus, and an in vitro ubiquitination assay revealed that the conserved RING finger domain is essential for its E3 ligase activity. We found that expression of LA4 can be induced by gravistimulation and that loss of LA4 function leads to defective shoot gravitropism caused by impaired asymmetric auxin redistribution upon gravistimulation. Genetic analysis demonstrated that LA4 acts in a distinct pathway from the starch biosynthesis regulators LA2 and LA3, which function in the starch-statolith-dependent pathway. Further genetic analysis showed that LA4 regulates shoot gravitropism and tiller angle by acting upstream of LA1 to mediate lateral auxin transport upon gravistimulation. Our studies reveal that LA4 regulates shoot gravitropism and tiller angle upstream of LA1 through a novel pathway independent of the LA2-LA3-mediated gravity-sensing mechanism, providing new insights into the rice tiller-angle regulatory network.

Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Low phosphorus promotes NSP1-NSP2 heterodimerization to enhance strigolactone biosynthesis and regulate shoot and root architecture in rice.

Phosphorus is an essential macronutrient for plant development and metabolism, and plants have evolved ingenious mechanisms to overcome phosphate (Pi) starvation. However, the molecular mechanisms underlying the regulation of shoot and root architecture by low phosphorus conditions and the coordinated utilization of Pi and nitrogen remain largely unclear. Here, we show that Nodulation Signaling Pathway 1 (NSP1) and NSP2 regulate rice tiller number by promoting the biosynthesis of strigolactones (SLs), a class of phytohormones with fundamental effects on plant architecture and environmental responses. We found that NSP1 and NSP2 are induced by Oryza sativa PHOSPHATE STARVATION RESPONSE2 (OsPHR2) in response to low-Pi stress and form a complex to directly bind the promoters of SL biosynthesis genes, thus markedly increasing SL biosynthesis in rice. Interestingly, the NSP1/2-SL signaling module represses the expression of CROWN ROOTLESS 1 (CRL1), a newly identified early SL-responsive gene in roots, to restrain lateral root density under Pi deficiency. We also demonstrated that GR244DO treatment under normal conditions inhibits the expression of OsNRTs and OsAMTs to suppress nitrogen absorption but enhances the expression of OsPTs to promote Pi absorption, thus facilitating the balance between nitrogen and phosphorus uptake in rice. Importantly, we found that NSP1p:NSP1 and NSP2p:NSP2 transgenic plants show improved agronomic traits and grain yield under low- and medium-phosphorus conditions. Taken together, these results revealed a novel regulatory mechanism of SL biosynthesis and signaling in response to Pi starvation, providing genetic resources for improving plant architecture and nutrient-use efficiency in low-Pi environments.

Essential Protein PHB2 and Its Regulatory Mechanisms in Cancer.

Prohibitins (PHBs) are a highly conserved class of proteins and have an essential role in transcription, epigenetic regulation, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism. Prohibitins form a heterodimeric complex, consisting of two proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2). They have been discovered to have crucial roles in regulating cancer and other metabolic diseases, functioning both together and independently. As there have been many previously published reviews on PHB1, this review focuses on the lesser studied prohibitin, PHB2. The role of PHB2 in cancer is controversial. In most human cancers, overexpressed PHB2 enhances tumor progression, while in some cancers, it suppresses tumor progression. In this review, we focus on (1) the history, family, and structure of prohibitins, (2) the essential location-dependent functions of PHB2, (3) dysfunction in cancer, and (4) the promising modulators to target PHB2. At the end, we discuss future directions and the clinical significance of this common essential gene in cancer.

Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice.

The sedentary lifestyle and refined food consumption significantly lead to obesity, type 2 diabetes, and related complications, which have become one of the major threats to global health. This incidence could be potentially reduced by daily foods rich in resistant starch (RS). However, it remains a challenge to breed high-RS rice varieties. Here, we reported a high-RS mutant rs4 with an RS content of ~10.8% in cooked rice. The genetic study revealed that the loss-of-function SSIIIb and SSIIIa together with a strong Wx allele in the background collaboratively contributed to the high-RS phenotype of the rs4 mutant. The increased RS contents in ssIIIa and ssIIIa ssIIIb mutants were associated with the increased amylose and lipid contents. SSIIIb and SSIIIa proteins were functionally redundant, whereas SSIIIb mainly functioned in leaves and SSIIIa largely in endosperm owing to their divergent tissue-specific expression patterns. Furthermore, we found that SSIII experienced duplication in different cereals, of which one SSIII paralog was mainly expressed in leaves and another in the endosperm. SSII but not SSIV showed a similar evolutionary pattern to SSIII. The copies of endosperm-expressed SSIII and SSII were associated with high total starch contents and low RS levels in the seeds of tested cereals, compared with low starch contents and high RS levels in tested dicots. These results provided critical genetic resources for breeding high-RS rice cultivars, and the evolutionary features of these genes may facilitate to generate high-RS varieties in different cereals.

A ciprofloxacin derivative with four mechanisms of action overcomes paclitaxel resistance in p53-mutant and MDR1 gene-expressing type II human endometrial cancer.

Dysfunction of the p53 gene and the presence of the MDR1 gene are associated with many malignant tumors including endometrial cancer and are responsible for cancer therapeutic resistance and poor survival. Thus, there is a critical need to devise novel combinatorial therapies with multiple mechanisms of action to overcome drug resistance. Here, we report a new ciprofloxacin derivative (CIP2b) tested either alone or in combination with taxanes against four human endometrial cancer cell lines. In vitro studies revealed that a combination of paclitaxel + CIP2b had synergistic cytotoxic effects against MDR1-expressing type-II human endometrial cancer cells with loss-of-function p53 (Hec50co LOFp53). Enhanced antitumor effects were confirmed by substantial increases in caspase-3 expression, cell population shifts toward the G2/M phase, and reduction of cdc2 phosphorylation. It was found that CIP2b targets multiple pathways including the inhibition of MDR1, topoisomerase I, and topoisomerase II, as well as enhancing the effects of paclitaxel (PTX) on microtubule assembly. In vivo treatment with the combination of PTX + CIP2b also led to significantly increased accumulation of PTX in tumors (compared to CIP2b alone) and reduction in tumor growth. Enhanced in vivo cytotoxic effects were confirmed by histological and immunohistochemical examination of the tumor tissues. Complete blood count and blood biochemistry data confirmed the absence of any apparent off-target toxicity. Thus, combination therapy involving PTX and CIP2b targeted multiple pathways and represents an approach that could result in improved tolerance and efficacy in patients with type-II endometrial cancer harboring the MDR1 gene and p53 mutations.

OsFTL12, a member of FT-like family, modulates the heading date and plant architecture by florigen repression complex in rice.

FLOWERING LOCUS T (FT), a florigen in Arabidopsis, plays critical roles in floral transition. Among 13 FT-like members in rice, OsFTL2 (Hd3a) and OsFTL3 (RFT1), two rice homologues of FT, have been well characterized to act as florigens to induce flowering under short-day (SD) and long-day (LD) conditions, respectively, but the functions of other rice FT-like members remain largely unclear. Here, we show that OsFTL12 plays an antagonistic function against Hd3a and RFT1 to modulate the heading date and plant architecture in rice. Unlike Hd3a and RFT1, OsFTL12 is not regulated by daylength and highly expressed in both SD and LD conditions, and delays the heading date under either SD or LD conditions. We further demonstrate that OsFTL12 interacts with GF14b and OsFD1, two key components of the florigen activation complex (FAC), to form the florigen repression complex (FRC) by competing with Hd3a for binding GF14b. Notably, OsFTL12-FRC can bind to the promoters of the floral identity genes OsMADS14 and OsMADS15 and suppress their expression. The osmads14 osmads15 double mutants could not develop panicles and showed erect leaves. Taken together, our results reveal that different FT-like members can fine-tune heading date and plant architecture by regulating the balance of FAC and FRC in rice.

Enhancing progestin therapy via HDAC inhibitors in endometrial cancer.

Uterine endometrial cancer (EC) incidence and deaths are on the rise. Hormone therapy, a traditional treatment regimen for this disease, uses progesterone and its synthetic analogue, progestin, to induce cell differentiation, apoptosis, and inhibition of invasion. This therapy is highly effective for progesterone receptor (PR) positive tumors in the short term. However, responsiveness decreases over time due to loss of PR expression; acquired resistance leads to treatment failure and poor prognosis. Primary resistance occurs in advanced, PR-negative tumors. Regardless, progestin therapy can be effective if the PR downregulation mechanism is reversed and if functional PR expression is restored. Using histone deacetylase inhibitors (HDACi), we inhibited cell proliferation in three EC cell lines and restored functional PR expression at the mRNA and protein levels. Two HDACi were tested using an endometrial xenograft tumor model: entinostat, an oral drug, and romidepsin, an IV drug. In vitro and in vivo studies support that entinostat decreased EC tumor growth, induced differentiation, and increased expression of the PR-targeted gene, PAEP. These findings supported the approval of a new NIH NCTN clinical trial, NRG-GY011, which concluded that dual treatment of MPA and entinostat, decreased expression of the proliferation marker, Ki67, but did not increase PR expression relative to single treatment with MPA in this short-term study. Therefore, a more potent HDACi, romidepsin, was investigated. Romidepsin treatment inhibited tumor growth and enhanced progestin treatment efficacy. More importantly, PR, PAEP, and KIAA1324 expressions were upregulated. Using a chromatin immunoprecipitation assay, we verified that HDACi can reverse PR downregulation mechanisms in mice models. Other potential drug efficacy markers, such as CD52, DLK1, GALNT9, and GNG2, were identified by transcriptome analysis and verified by q-PCR. Many of the upregulated drug efficacy markers predict favorable patient outcomes, while downregulated genes predict worse survival. Here, our current data suggests that romidepsin is a more potent HDACi that has the potential to achieve more robust upregulation of PR expression and may be a more promising candidate for future clinical trials.

Protocol for genome editing in wild allotetraploid rice Oryza alta.

We present a protocol for Agrobacterium-mediated genetic transformation and genome editing in wild allotetraploid rice Oryza alta. We detail steps to induce embryogenic calli from mature seeds and co-cultivate with Agrobacterium after infection. We further describe how to select transformed cells that proliferated from infected calli based on hygromycin resistance, resulting in regeneration of transformed plantlets with multiple edited alleles. Edited lines produced via this protocol can be used for gene functions studies and de novo domestication of O. alta. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).

Utilizing an Endogenous Progesterone Receptor Reporter Gene for Drug Screening and Mechanistic Study in Endometrial Cancer.

Expression of progesterone receptor (PR) is a favorable prognostic marker for multiple solid tumors. However, PR expression is reduced or lost in malignant tumors. Thus, monitoring and restoring functional PR expression is important in order to sensitize tumor cells to progesterone therapy in endometrial cancer. We developed stable PR reporter gene containing endometrial cancer cell lines monitoring the endogenous PR expression by inserting mCherry and hygromycin resistant gene at the endogenous PR gene locus by CRISPR/Cas9-mediated genome editing technique. This allows efficient, real-time monitoring of PR expression in its native epigenetic landscape. Reporter gene expression faithfully reflects and amplifies PR expression following treatment with drugs known to induce PR expression. Small molecular PR inducers have been identified from the FDA-approved 1018 drug library and tested for their ability to restore PR expression. Additionally, several candidate PR repressors have been identified by screening the genome-wide CRISPR knockout (GeCKO) library. This novel endogenous PR reporter gene system facilitates the discovery of a new treatment strategy to enhance PR expression and further sensitize progestin therapy in endometrial cancer. These tools provide a systematic, unbiased approach for monitoring target gene expression, allowing for novel drug discovery and mechanistic exploration.

Chilling-induced phosphorylation of IPA1 by OsSAPK6 activates chilling tolerance responses in rice.

Chilling is a major abiotic stress harming rice development and productivity. The C-REPEAT BINDING FACTOR (CBF)-dependent transcriptional regulatory pathway plays a central role in cold stress and acclimation in Arabidopsis. In rice, several genes have been reported in conferring chilling tolerance, however, the chilling signaling in rice remains largely unknown. Here, we report the chilling-induced OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 6 (OsSAPK6)-IDEAL PLANT ARCHITECTURE 1 (IPA1)-OsCBF3 signal pathway in rice. Under chilling stress, OsSAPK6 could phosphorylate IPA1 and increase its stability. In turn, IPA1 could directly bind to the GTAC motif on the OsCBF3 promoter to elevate its expression. Genetic evidence showed that OsSAPK6, IPA1 and OsCBF3 were all positive regulators of rice chilling tolerance. The function of OsSAPK6 in chilling tolerance depended on IPA1, and overexpression of OsCBF3 could rescue the chilling-sensitive phenotype of ipa1 loss-of-function mutant. Moreover, the natural gain-of-function allele ipa1-2D could simultaneously enhance seedling chilling tolerance and increase grain yield. Taken together, our results revealed a chilling-induced OsSAPK6-IPA1-OsCBF signal cascade in rice, which shed new lights on chilling stress-tolerant rice breeding.

OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice.

Salt stress adversely affects plant growth, development, and crop yield. Rice (Oryza sativa L.) is one of the most salt-sensitive cereal crops, especially at the early seedling stage. Mitogen-activated protein kinase (MAPK/MPK) cascades have been shown to play critical roles in salt response in Arabidopsis. However, the roles of the MPK cascade signaling in rice salt response and substrates of OsMPK remain largely unknown. Here, we report that the salt-induced OsMPK4-Ideal Plant Architecture 1 (IPA1) signaling pathway regulates the salt tolerance in rice. Under salt stress, OsMPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180, leading to degradation of IPA1. Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice, whereas OsMPK4 promotes salt response in an IPA1-dependent manner. Taken together, our results uncover an OsMPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.

An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro.

Multisubunit SKP1/Cullin1/F-box (SCF) E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes. Detecting E3 ligase activity in vitro is important for exploring the molecular mechanism of protein ubiquitination. However, in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve, especially in plants, mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs. In this study, we characterized components of the rice SCFDWARF3 (SCFD3) E3 ligase, screened the coordinated E2, and reconstituted active SCFD3 E3 ligase in vitro. We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1 (eSCR) protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53, which is the key transcriptional repressor in strigolactone signaling. Finally, we replaced D3 with other F-box proteins from rice and humans and reconstituted active eSCF E3 ligases, including eSCFGID2, eSCFFBXL18, and eSCFCDC4 E3 ligases. Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins, providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size.

Crop genetic improvement requires balancing complex tradeoffs caused by gene pleiotropy and linkage drags, as exemplified by IPA1 (Ideal Plant Architecture 1), a typical pleiotropic gene in rice that increases grains per panicle but reduces tillers. In this study, we identified a 54-base pair cis-regulatory region in IPA1 via a tiling-deletion-based CRISPR-Cas9 screen that, when deleted, resolves the tradeoff between grains per panicle and tiller number, leading to substantially enhanced grain yield per plant. Mechanistic studies revealed that the deleted fragment is a target site for the transcription factor An-1 to repress IPA1 expression in panicles and roots. Targeting gene regulatory regions should help dissect tradeoff effects and provide a rich source of targets for breeding complementary beneficial traits.