An ancestral role for 3-KETOACYL-COA SYNTHASE3 as a negative regulator of plant cuticular wax synthesis.

The plant cuticle, a structure primarily composed of wax and cutin, forms a continuous coating over most aerial plant surfaces. The cuticle plays important roles in plant tolerance to environmental stress, including stress imposed by drought. Some members of the 3-KETOACYL-COA SYNTHASE (KCS) family are known to act as metabolic enzymes involved in cuticular wax production. Here we report that Arabidopsis (Arabidopsis thaliana) KCS3, which was previously shown to lack canonical catalytic activity, instead functions as a negative regulator of wax metabolism by reducing the enzymatic activity of KCS6, a key KCS involved in wax production. We demonstrate that the role of KCS3 in regulating KCS6 activity involves physical interactions between specific subunits of the fatty acid elongation complex and is essential for maintaining wax homeostasis. We also show that the role of the KCS3-KCS6 module in regulating wax synthesis is highly conserved across diverse plant taxa from Arabidopsis to the moss Physcomitrium patens, pointing to a critical ancient and basal function of this module in finely regulating wax synthesis.

The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers.

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.

ENHANCED DISEASE SUSCEPTIBILITY 1 promotes hydrogen peroxide scavenging to enhance rice thermotolerance.

Heat stress is a major factor limiting the production and geographic distribution of rice (Oryza sativa), and breeding rice varieties with tolerance to heat stress is of immense importance. Although extensive studies have revealed that reactive oxygen species (ROS) play a critical role in rice acclimation to heat stress, the molecular basis of rice controlling ROS homeostasis remains largely unclear. In this study, we discovered a novel heat-stress-responsive strategy that orchestrates ROS homeostasis centering on an immune activator, rice ENHANCED DISEASE SUSCEPTIBILITY 1 (OsEDS1). OsEDS1, which confers heat stress tolerance, promotes hydrogen peroxide (H2O2) scavenging by stimulating catalase activity through the OsEDS1-catalase association. The loss-of-function mutation in OsEDS1 causes increased sensitivity to heat stress, whereas the overexpression of OsEDS1 enhances thermotolerance. Furthermore, overexpression lines greatly improved rice tolerance to heat stress during the reproductive stage, which was associated with substantially increased seed setting, grain weight, and plant yield. Rice CATALASE C (OsCATC), whose activity is promoted by OsEDS1, degrades H2O2 to activate rice heat stress tolerance. Our findings greatly expand our understanding of heat stress responses in rice. We reveal a molecular framework that promotes heat tolerance through ROS homeostasis regulation, suggesting a theoretical basis and providing genetic resources for breeding heat-tolerant rice varieties.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 13 (SLP13) together with SPL9 redundantly regulates wax biosynthesis under drought stress.

Wax biosynthesis is closely controlled by many regulators under different environmental conditions. We have previously shown that the module miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)-DEWAX is involved in the diurnal regulation of wax production; however, it was not determined whether other SPLs are also involved in wax synthesis. Here, we report that SPL13 also regulates drought-induced wax production, by directly and indirectly affecting the expression of the two wax biosynthesis genes ECERIFERUM1 (CER1) and CER4, respectively. In addition, we show that SPL13 together with SPL9 redundantly regulates wax accumulation under both normal and drought stress conditions, and that simultaneous mutation of both genes additively increases cuticle permeability and decreases drought tolerance. However, in contrast to SPL9, SPL13 does not seem to participate in the DEWAX-mediated diurnal regulation of wax production.

OsMBF1a Facilitates Seed Germination by Regulating Biosynthesis of Gibberellic Acid and Abscisic Acid in Rice.

Seed germination is a pivotal stage in the plant life cycle, orchestrated by a myriad of internal and external factors, notably plant hormones. The underlying molecular mechanisms governing rice seed germination remain largely unelucidated. Herein, we uncover OsMBF1a as a crucial regulatory factor that employs a dual strategy to promote seed germination: positively activating genes involved in gibberellin (GA) biosynthesis pathways, while negatively regulating key genes responsible for abscisic acid (ABA) synthesis. Furthermore, OsMBF1a modulates the endogenous levels of ABA and GA in rice seeds, reinforcing its central role in the germination process. The expression of ZmMBF1a and ZmMBF1b, the homologous genes in maize, in rice seeds similarly affects germination, indicating the conserved functionality of MBF1 family genes in regulating seed germination. This study provides novel insights into the molecular mechanisms underlying rice seed germination and underscores the significance of MBF1 family genes in plant growth and development.

Regulatory network of rice in response to heat stress and its potential application in breeding strategy.

The rapid development of global industrialization has led to serious environmental problems, among which global warming has become one of the major concerns. The gradual rise in global temperature resulted in the loss of food production, and hence a serious threat to world food security. Rice is the main crop for approximately half of the world's population, and its geographic distribution, yield, and quality are frequently reduced due to elevated temperature stress, and breeding rice varieties with tolerance to heat stress is of immense significance. Therefore, it is critical to study the molecular mechanism of rice in response to heat stress. In the last decades, large amounts of studies have been conducted focusing on rice heat stress response. Valuable information has been obtained, which not only sheds light on the regulatory network underlying this physiological process but also provides some candidate genes for improved heat tolerance breeding in rice. In this review, we summarized the studies in this field. Hopefully, it will provide some new insights into the mechanisms of rice under high temperature stress and clues for future engineering breeding of improved heat tolerance rice.

Mutations on ent-kaurene oxidase 1 encoding gene attenuate its enzyme activity of catalyzing the reaction from ent-kaurene to ent-kaurenoic acid and lead to delayed germination in rice.

Rice seed germination is a critical step that determines its entire life circle, with seeds failing to germinate or pre-harvest sprouting both reduce grain yield. Nevertheless, the mechanisms underlying this complex biological event remain unclear. Previously, gibberellin has been shown to promote seed germination. In this study, a delayed seed germination rice mutant was obtained through screening of the EMS induced mutants. Besides of delayed germination, it also shows semi-dwarfism phenotype, which could be recovered by exogenous GA. Through re-sequencing on the mutant, wild-type and their F2 populations, we identified two continuous mutated sites on ent-kaurene oxidase 1 (OsKO1) gene, which result in the conversion from Thr to Met in the cytochrome P450 domain. Genetic complementary analysis and enzyme assay verified that the mutations in OsKO1 gene block the biosynthesis of GA and result in the defect phenotypes. Further analyses proved that OsKO1 could catalyze the reaction from ent-kaurene into ent-kaurenoic acid in GA biosynthesis mainly at seed germination and seedling stages, and the mutations decrease its activity to catalyze the step from ent-kaurenol to ent-kaurenoic acid in this reaction. Transcriptomic and proteomic data indicate that the defect on GA biosynthesis decreases its ability to mobilize starch and attenuate ABA signaling, therefore delay the germination process. The results provide some new insights into both GA biosynthesis and seed germination regulatory pathway in rice.

Advances of Apetala2/Ethylene Response Factors in Regulating Development and Stress Response in Maize.

Apetala2/ethylene response factor (AP2/ERF) is one of the largest families of transcription factors, regulating growth, development, and stress response in plants. Several studies have been conducted to clarify their roles in Arabidopsis and rice. However, less research has been carried out on maize. In this review, we systematically identified the AP2/ERFs in the maize genome and summarized the research progress related to AP2/ERF genes. The potential roles were predicted from rice homologs based on phylogenetic and collinear analysis. The putative regulatory interactions mediated by maize AP2/ERFs were discovered according to integrated data sources, implying that they involved complex networks in biological activities. This will facilitate the functional assignment of AP2/ERFs and their applications in breeding strategy.

A stress-associated protein OsSAP8 modulates gibberellic acid biosynthesis by reducing the promotive effect of transcription factor OsbZIP58 on OsKO2.

Gibberellic acid (GA) is a vital phytohormone for plant growth and development. GA biosynthesis is a complex pathway regulated by various transcription factors. Here we report a stress-associated protein 8 (OsSAP8), negatively involved in GA biosynthesis. Overexpression of OsSAP8 in rice resulted in a semi-dwarfism phenotype and reduced endogenous GA3 content. In contrast, an OsSAP8 knockout mutant exhibited higher endogenous GA3 content and slightly increased plant height. Sub-cellular localization analysis of OsSAP8 showed that it could enter the nucleus. Based on electrophoretic mobility shift assay and yeast one hybrid experiments, OsSAP8 was found to bind to the cis-acting regulatory element GADOWNAT of ent-kaurene oxidases (KO2, KO3, KO5). The results from dual-luciferase reporter assays showed that OsSAP8 does not activate LUC reporter gene expression. However, it could interact with basic leucine zipper 58 (OsbZIP58), which has strong transcriptional activation potential on OsKO2. Moreover, the interaction between OsSAP8, rice lesion simulating disease 1-like 1 (OsLOL1), and OsbZIP58 could reduce the promotive effect of transcription factor OsbZIP58 on OsKO2. These results provide some new insights on the regulation of GA biosynthesis in rice.

Studies on Lotus Genomics and the Contribution to Its Breeding.

Lotus (Nelumbo nucifera), under the Nelumbonaceae family, is one of the relict plants possessing important scientific research and economic values. Because of this, much attention has been paid to this species on both its biology and breeding among the scientific community. In the last decade, the genome of lotus has been sequenced, and several high-quality genome assemblies are available, which have significantly facilitated functional genomics studies in lotus. Meanwhile, re-sequencing of the natural and genetic populations along with different levels of omics studies have not only helped to classify the germplasm resources but also to identify the domestication of selected regions and genes controlling different horticultural traits. This review summarizes the latest progress of all these studies on lotus and discusses their potential application in lotus breeding.

Quantitative ubiquitylomics approach for characterizing the dynamic change and extensive modulation of ubiquitylation in rice seed germination.

During seed germination, cells embark on extensive post-transcriptional and post-translational modifications (PTM), providing a perfect platform to study these events in embryo rebooting from relative quiescenct to highly active state. PR-619, a deubiquitylase inhibitor, delayed the rice seed germination and resulted in the accumulation of ubiquitylated proteins, which indicated the protein ubiquitylation is involved in this process. Using the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry, a list of 2576 lysine ubiquitylated (Kub) sites in 1171 proteins was compiled for rice embryos at 0, 12 and 24 h after imbibition (HAI). Of these, the abundance of 1419 Kub sites in 777 proteins changed significantly. Most of them substantially increased within the first 12 HAI, which is similar to the dynamic state previously observed for protein phosphorylation, implying that the first 12 HAI are essential for subsequent switch during rice seed germination. We also quantitatively analyzed the embryo proteome in these samples. Generally, a specific protein's abundance in the ubiquitylome was uncorrelated to that in the proteome. The differentially ubiquitinated proteins were greatly enriched in the categories of protein processing, DNA and RNA processing/regulation related, signaling, and transport. The DiGly footprint of the Kub sites was significantly reduced on K48, a linkage typically associated with proteasome-mediated degradation. These observations suggest ubiquitylation may modulate the protein function more than providing 26S degradation signals in the early stage of rice seed germination. Revealing this comprehensive ubiquitylome greatly increases our understanding of this critical PTM during seed germination.

Genome-wide analysis of AP2/ERF superfamily in lotus (Nelumbo nucifera) and the association between NnADAP and rhizome morphology.

BACKGROUND: The AP2/ERF family is widely present in plants and plays a crucial regulatory role in plant growth and development. As an essential aquatic horticultural model plant, lotus has an increasingly prominent economic and research value. RESULTS: We have identified and analysed the AP2/ERF gene family in the lotus. Initially, 121 AP2/ERF family genes were identified. By analysing their gene distribution and protein structure, and their expression patterns during the development of lotus rhizome, combined with previous studies, we obtained an SNP (megascaffold_20:3578539) associated with lotus rhizome phenotype. This SNP was in the NnADAP gene of the AP2 subfamily, and the changes in SNP (C/T) caused amino acid conversion (proline/leucine). We constructed a population of 95 lotus varieties for SNP verification. Through population typing experiments, we found that the group with SNP CC had significantly larger lotus rhizome and higher soluble sugar content among the population. CONCLUSIONS: In conclusion, we speculate that the alteration of the SNP in the NnADAP can affect the size and sugar content of the lotus rhizome.

Biased allelic expression in tissues of F1 hybrids between tropical and temperate lotus (Nelumbo nuicfera).

KEY MESSAGE: The genome-wide allele-specific expression in F1 hybrids from the cross of tropical and temperate lotus unveils how cis-regulatory divergences affect genes in key pathways related to ecotypic divergence. Genetic variation, particularly cis-regulatory variation, plays a crucial role in phenotypic variation and adaptive evolution in plants. Temperate and tropical lotus, the two ecotypes of Nelumbo nucifera, show distinction in the degree of rhizome enlargement, which is associated with winter dormancy. To understand the roles of genome-wide cis-regulatory divergences on adaptive evolution of temperate and tropical lotus (Nelumbo nucifera), here we performed allele-specific expression (ASE) analyses on the tissues including flowers, leaves and rhizome from F1 hybrids of tropical and temperate lotus. For all investigated tissues in F1s, about 36% of genes showed ASE and about 3% of genes showed strong consistent ASE. Most of ASEs were biased towards the tropical parent in all surveyed samples, indicating that the tropical genome might be dominant over the temperate genome in gene expression of tissues from their F1 hybrids. We found that promoter sequences with similar allelic expression are more conserved than genes with significant or conditional ASE, suggesting the cis-regulatory sequence divergence underlie the allelic expression bias. We further uncovered biased genes being related to phenotypic differentiation between two lotus ecotypes, especially metabolic and phytohormone-related pathways in the rhizome. Overall, our study provides a global landscape of cis-regulatory variations between two lotus ecotypes and highlights their roles in rhizome growth variation for the climatic adaptation.

A CONSTANS-LIKE gene of Nelumbo nucifera could promote potato tuberization.

MAIN CONCLUSION: CONSTANS-LIKE 5 of Nelumbo nucifera is capable of promoting potato tuberization through CONSTANS-FLOWERING LOCUS T and gibberellin signaling pathways with a probable association with lotus rhizome enlargement. Lotus (Nelumbo nucifera) is an aquatic plant that is affiliated to the Nelumbonaceace family. It is widely used as an ornamental, vegetable, and medicinal herb with its rhizome being a popular vegetable. To explore the molecular mechanism underlying its rhizome enlargement, we conducted a systematic analysis on the CONSTANS-LIKE (COL) gene family, with the results, indicating that this gene plays a role in regulating potato tuber expansion. These analyses included phylogenetic relationships, gene structure, and expressional patterns of lotus COL family genes. Based on these analyses, NnCOL5 was selected for further study on its potential function in lotus rhizome formation. NnCOL5 was shown to be located in the nucleus, and its expression was positively associated with the enlargement of lotus rhizome. Besides, the overexpression of NnCOL5 in potato led to increased tuber weight and starch content under short-day conditions without changing the number of tubers. Further analysis suggested that the observed tuber changes might be mediated by affecting the expression of genes in CO-FT and GA signaling pathways. These results provide valuable insight in understanding the functions of COL gene as well as the enlargement of lotus rhizome.

Improving the Genome Annotation of Rhizoctonia solani Using Proteogenomics.

Background: Rhizoctonia solani is a pathogenic fungus that causes serious diseases in many crops, including rice, wheat, and soybeans. In crop production, it is very important to understand the pathogenicity of this fungus, which is still elusive. It might be helpful to comprehensively understand its genomic information using different genome annotation strategies. Methods: Aiming to improve the genome annotation of R. solani, we performed a proteogenomic study based on the existing data. Based on our study, a total of 1060 newly identified genes, 36 revised genes, 139 single amino acid variants (SAAVs), 8 alternative splicing genes, and diverse post-translational modifications (PTMs) events were identified in R. solani AG3. Further functional annotation on these 1060 newly identified genes was performed through homology analysis with its 5 closest relative fungi. Results: Based on this, 2 novel candidate pathogenic genes, which might be associated with pathogen-host interaction, were discovered. In addition, in order to increase the reliability and novelty of the newly identified genes in R. solani AG3, 1060 newly identified genes were compared with the newly published available R. solani genome sequences of AG1, AG2, AG4, AG5, AG6, and AG8. There are 490 homologous sequences. We combined the proteogenomic results with the genome alignment results and finally identified 570 novel genes in R. solani. Conclusion: These findings extended R. solani genome annotation and provided a wealth of resources for research on R. solani.

Metabolomics Analyses of Cotyledon and Plumule Showing the Potential Domestic Selection in Lotus Breeding.

Lotus (Nelumbo nucifera) seeds are widely consumed as functional food or herbal medicine, of which cotyledon (CL) is the main edible part, and lotus plumule (LP) is commonly utilized in traditional Chinese medicine. However, few studies have been conducted to investigate the chemical components of CL and LP in dry lotus seeds, not to mention the comparison between wild and domesticated varieties. In this study, a widely targeted metabolomics approach based on Ultra Performance Liquid Chromatography-electrospray ionization-Tandem mass spectrometry (UPLC-ESI-MS/MS) was utilized to analyze the metabolites in CL and LP of China Antique ("CA", a wild variety) and Jianxuan-17 ("JX", a popular cultivar). A total of 402 metabolites were identified, which included flavonoids (23.08% to 27.84%), amino acids and derivatives (14.18-16.57%), phenolic acids (11.49-12.63%), and lipids (9.14-10.95%). These metabolites were classified into ten clusters based on their organ or cultivar-specific characters. Most of these metabolites were more abundant in LP than in CL for both varieties, except for metabolites belonging to organic acids and lipids. The analysis of differentially accumulated metabolites (DAMs) demonstrated that more than 25% of metabolites detected in our study were DAMs in CL and LP comparing "JX" with "CA", most of which were less abundant in "JX", including 35 flavonoids in LP, 23 amino acids and derivatives in CL, 7 alkaloids in CL, and 10 nucleotides and derivatives in LP, whereas all of 11 differentially accumulated lipids in LP were more abundant in "JX". Together with the fact that the seed yield of "JX" is much higher than that of "CA", these results indicated that abundant metabolites, especially the functional secondary metabolites (mainly flavonoids and alkaloids), were lost during the process of breeding selection.

Genome-wide identification of MADS-box gene family in sacred lotus (Nelumbo nucifera) identifies a SEPALLATA homolog gene involved in floral development.

BACKGROUND: Sacred lotus (Nelumbo nucifera) is a vital perennial aquatic ornamental plant. Its flower shape determines the horticultural and ornamental values. However, the mechanisms underlying lotus flower development are still elusive. MADS-box transcription factors are crucial in various features of plant development, especially in floral organogenesis and specification. It is still unknown how the MADS-box transcription factors regulate the floral organogenesis in lotus. RESULTS: To obtain a comprehensive insight into the functions of MADS-box genes in sacred lotus flower development, we systematically characterized members of this gene family based on the available genome information. A total of 44 MADS-box genes were identified, of which 16 type I and 28 type II genes were categorized based on the phylogenetic analysis. Furthermore, the structure of MADS-box genes and their expressional patterns were also systematically analyzed. Additionally, subcellular localization analysis showed that they are mainly localized in the nucleus, of which a SEPALLATA3 (SEP3) homolog NnMADS14 was proven to be involved in the floral organogenesis. CONCLUSION: These results provide some fundamental information about the MADS-box gene family and their functions, which might be helpful in not only understanding the mechanisms of floral organogenesis but also breeding of high ornamental value cultivars in lotus.

Integrated Omics Analyses Identify Key Pathways Involved in Petiole Rigidity Formation in Sacred Lotus.

Sacred lotus (Nelumbo nucifera Gaertn.) is a relic aquatic plant with two types of leaves, which have distinct rigidity of petioles. Here we assess the difference from anatomic structure to the expression of genes and proteins in two petioles types, and identify key pathways involved in petiole rigidity formation in sacred lotus. Anatomically, great variation between the petioles of floating and vertical leaves were observed. The number of collenchyma cells and thickness of xylem vessel cell wall was higher in the initial vertical leaves' petiole (IVP) compared to the initial floating leaves' petiole (IFP). Among quantified transcripts and proteins, 1021 and 401 transcripts presented 2-fold expression increment (named DEGs, genes differentially expressed between IFP and IVP) in IFP and IVP, 421 and 483 proteins exhibited 1.5-fold expression increment (named DEPs, proteins differentially expressed between IFP and IVP) in IFP and IVP, respectively. Gene function and pathway enrichment analysis displayed that DEGs and DEPs were significantly enriched in cell wall biosynthesis and lignin biosynthesis. In consistent with genes and proteins expressions in lignin biosynthesis, the contents of lignin monomers precursors were significantly different in IFP and IVP. These results enable us to understand lotus petioles rigidity formation better and provide valuable candidate genes information on further investigation.

Advances on Plant Ubiquitylome-From Mechanism to Application.

Post-translational modifications (PTMs) of proteins enable modulation of their structure, function, localization and turnover. To date, over 660 PTMs have been reported, among which, reversible PTMs are regarded as the key players in cellular signaling. Signaling mediated by PTMs is faster than re-initiation of gene expression, which may result in a faster response that is particularly crucial for plants due to their sessile nature. Ubiquitylation has been widely reported to be involved in many aspects of plant growth and development and it is largely determined by its target protein. It is therefore of high interest to explore new ubiquitylated proteins/sites to obtain new insights into its mechanism and functions. In the last decades, extensive protein profiling of ubiquitylation has been achieved in different plants due to the advancement in ubiquitylated proteins (or peptides) affinity and mass spectrometry techniques. This obtained information on a large number of ubiquitylated proteins/sites helps crack the mechanism of ubiquitylation in plants. In this review, we have summarized the latest advances in protein ubiquitylation to gain comprehensive and updated knowledge in this field. Besides, the current and future challenges and barriers are also reviewed and discussed.

Proteomic analysis showing the signaling pathways involved in the rhizome enlargement process in Nelumbo nucifera.

BACKGROUND: Rhizome is the storage underground stem of lotus (Nelumbo nucifera), which is enlarged before winter season and could be used for asexual propagation. In addition, the enlarged rhizome is a nutritional vegetable with abundant starch, proteins, and vitamins. Enlargement of lotus rhizome is not only significance for itself to survive from the cold winter, but also important for its economic value. RESULTS: To explore the mechanism underlying its enlargement, integrative analyses of morphology, physiology and proteomics were conducted on the rhizome at stolon, middle, and enlarged stages. Morphological observation and physiological analyses showed that rhizomes were gradually enlarged during this process, in which the starch accumulation was also initiated. Quantitative proteomic analysis on the rhizomes at these three stages identified 302 stage-specific proteins (SSPs) and 172 differently expressed proteins (DEPs), based on which GO and KEGG enrichment analyses were conducted. The results indicated that light and auxin signal might be transduced through secondary messenger Ca2+, and play important roles in lotus rhizome enlargement. CONCLUSION: These results will provide new insights into understanding the mechanism of lotus rhizome enlargement. Meanwhile, some candidate genes might be useful for further studies on this process, as well as breeding of rhizome lotus.