Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Comparative transcriptome analysis of pollen and anther wall reveals novel insights into the regulatory mechanisms underlying anther wall development and its dehiscence in rice.

To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for the following two tissues: the anther wall and pollen at the anthesis stage. With the anatomical meta-expression data, in addition to these tissues, the differentially expressed genes (DEGs) between the two tissues were further refined to identify 1,717 pollen-preferred genes and 534 anther wall-preferred genes. A GUS transgenic line and RT-qPCR analysis for anther wall-preferred genes supported the fidelity of our gene candidates for further analysis. The refined DEGs were functionally classified through Gene Ontology (GO) enrichment and MapMan analyses. Through the analysis of cis-acting elements and alternative splicing variants, we also suggest the feature of regulatory sequences in promoter regions for anther wall-preferred expression and provide information of the unique splicing variants in anther wall. Subsequently, it was found that hormone signaling and the resulting transcriptional regulation pathways may play an important role in anther dehiscence and anther wall development. Our results could provide useful insights into future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

Impact of Ecklonia stolonifera extract on in vitro ruminal fermentation characteristics, methanogenesis, and microbial populations.

OBJECTIVE: This study was conducted to evaluate the effects of E. stolonifera extract addition on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations. METHODS: Cannulated Holstein cows (450 ± 30 kg) consuming timothy hay and a commercial concentrate (60:40, w/w) twice daily (09:00 and 17:00) at 2% of body weight with free access to water and mineral block were used as rumen fluid donors. In vitro fermentation experiment, with timothy hay as substrate, was conducted for up to 72 h, with E. stolonifera extract added to achieve final concentration 1, 3 and 5% on timothy hay basis. RESULTS: Administration of E. stolonifera extract to a ruminant fluid-artificial saliva mixture in vitro increased the total gas production. Unexpectedly, E. stolonifera extracts appeared to increase both methane emissions and hydrogen production, which contrasts with previous observations with brown algae extracts used under in vitro fermentation conditions. Interestingly, real-time polymerase chain reaction indicated that as compared with the untreated control the ciliate-associated methanogen and Fibrobacter succinogenes populations decreased, whereas the Ruminococcus flavefaciens population increased as a result of E. stolonifera extract supplementation. CONCLUSIONS: E. stolonifera showed no detrimental effect on rumen fermentation characteristics and microbial population. Through these results E. stolonifera has potential as a viable feed supplement to ruminants.

Effects of Medicinal Herb Extracts on In vitro Ruminal Methanogenesis, Microbe Diversity and Fermentation System.

This study was aimed to evaluate the in vitro effects of medicinal herb extracts (MHEs) on ruminal fermentation characteristics and the inhibition of protozoa to reduce methane production in the rumen. A fistulated Hanwoo was used as a donor of rumen fluid. The MHEs (T1, Veratrum patulum; T2, Iris ensata var. spontanea; T3, Arisaema ringens; T4, Carduus crispus; T5, Pueraria thunbergiana) were added to the in vitro fermentation bottles containing the rumen fluid and medium. Total volatile fatty acid (tVFA), total gas production, gas profiles, and the ruminal microbe communities were measured. The tVFA concentration was increased or decreased as compared to the control, and there was a significant (p<0.05) difference after 24 h incubation. pH and ruminal disappearance of dry matter did not show significant difference. As the in vitro ruminal fermentation progressed, total gas production in added MHEs was increased, while the methane production was decreased compared to the control. In particular, Arisaema ringens extract led to decrease methane production by more than 43%. In addition, the result of real-time polymerase chain reaction indicted that the protozoa population in all added MHEs decreased more than that of the control. In conclusion, the results of this study indicated that MHEs could have properties that decrease ruminal methanogenesis by inhibiting protozoa species and might be promising feed additives for ruminants.

Phosphorylation of hypothalamic AMPK on serine(485/491) related to sustained weight loss by alpha-lipoic acid in mice treated with olanzapine.

RATIONALE: Alpha-lipoic acid (ALA) was shown to suppress atypical antipsychotic drug (AAPD)-induced weight gain. However, its mode of action has remained unidentified. OBJECTIVE: We aimed to identify mechanisms underlying anti-obesity effects of ALA in mice treated with olanzapine. METHODS: We compared body weight and food intake among vehicle-, olanzapine-, and olanzapine plus ALA-treated mice, and measured hypothalamic AMP-activated protein kinase (AMPK) activity by detecting levels of Thr(172) and Ser(485/491) phosphorylation, which indicate activation and inhibition of AMPK, respectively. RESULTS: Body weights were increased by olanzapine in parallel with increased levels of Thr(172) phosphorylation of hypothalamic AMPK. Initially increased rate of weight gain was diminished as Thr(172) phosphorylation levels were decreased to control levels after 10 days of olanzapine treatment. ALA successfully not only prevented olanzapine-induced weight gain but also induced additional weight loss even relative to control levels throughout the treatment period. During the initial stage, ALA's action was indicated by both suppression of olanzapine-induced Thr(172) phosphorylation and an increase in Ser(485/491) phosphorylation levels. However, in the later stage when no more increases in Thr(172) phosphorylation and weight gain by olanzapine were observed, ALA's action was only indicated by increased levels of Ser(485/491) phosphorylation. CONCLUSIONS: Our data suggest that anti-obesity effects of ALA may be related to modulation of both Ser(485/491) phosphorylation and Thr(172) phosphorylation of hypothalamic AMPK, while olanzapine-induced weight gain may be only associated with increase in Thr(172) phosphorylation. This might be an important mechanistic clue for the future development of anti-obesity drugs beyond control of AAPD-induced weight gain.

Cerebroprotective effects of red ginseng extract pretreatment against ischemia-induced oxidative stress and apoptosis.

Panax ginseng C.A. Meyer has been traditionally used as a medicinal plant and has beneficial effects due to pharmacological properties. Although ginseng is thought to be protective under abnormal conditions, the effects of pretreatment with red ginseng (RG) extract on ischemic stroke have not been fully elucidated. We investigated the protective effects of RG extract after focal cerebral ischemia in mice. Crude RG extract (360 mg/kg) was administered intraperitoneally for 2 weeks. Mice were then subjected to occlusion of the middle cerebral artery for 1 hour, followed by reperfusion for 4 and 24 hours. Pretreatment with RG extract followed by ischemia/reperfusion (I/R) resulted in significant reduction of oxidized hydroethidine signals in ischemic areas. At 4 and 24 hours after I/R, the number of 8-hydroxyguanosine and apoptosis signal-regulating kinase 1 (ASK1)-positive cells decreased in the ischemic penumbra as seen using immunofluorescent staining. Western blotting showed that RG efficiently attenuated the protein levels of activated ASK1 in the ischemic penumbra. Consequently, DNA fragmentation and the infarct volume were reduced by RG extract pretreatment 24 hours after I/R. Also, RG extract resulted in better performance in rotarod test after I/R. Thus, RG pretreatment demonstrates a protective effect at suppressing ischemia-induced oxidative stress and apoptosis in ischemic lesions. Pretreatment with crude RG extract may be an effective strategy for preventing brain injury after an ischemic stroke.