SPL13 together with SPL9 redundantly regulates wax biosynthesis upon drought stress.

Wax biosynthesis is strictly regulated by many regulators under different environmental conditions. Our previous study showed that the regulation module miR156/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)/DEWAX is identified to be involved in the diurnal regulation of wax production, however, it was unknown if other SPLs are also involved in the wax synthesis. Here, we reported that SPL13 regulates drought-induced wax production as well. Moreover, its regulatory role directly or indirectly affects the expression of two wax biosynthesis genes CER1 and CER4. Further study showed that SPL13 together with SPL9 redundantly regulated the wax accumulation upon either normal conditions or drought stress, simultaneous mutation of both genes additively enhanced cuticle permeability and decreased the drought tolerance. However, different from SPL9, SPL13 seemed not to participate in the DEWAX-mediated diurnal regulation of wax production.

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.

Exploring domestication pattern in lotus: insights from dispensable genome assembly.

Lotus (Nelumbo nucifera Gaertn.), an important aquatic plant in horticulture and ecosystems, has been cultivated for more than 7000 years and domesticated into three different subgroups: flower lotus, rhizome lotus, and seed lotus. To explore the domesticated regions of each subgroup, re-sequencing data of 371 lotus accessions collected from the public database were aligned to the genome of 'China-Antique (CA)'. Unmapped reads were used to build the dispensable genome of each subgroup using a metagenome-like assembly strategy. More than 27 Mb of the dispensable genome in these three subgroups and the wild group was assembled, of which 11,761 genes were annotated. Some of the contigs in the dispensable genome were similar to the genomic segments of other lotus accessions other than 'CA'. The annotated genes in each subgroup played essential roles in specific developmental processes. Dissection of selective signals in three cultivated subgroups also demonstrated that subgroup-specific metabolic pathways, such as the brassinosteroids metabolism enrichment in FL, associated with these selected genes in each subgroup and the contigs in dispensable genome nearly located in the domesticated regions of each subgroup, respectively. Our data presented a valuable resource for facilitating lotus genomic studies, complemented the helpful information to the reference genome, and shed light on the selective signals of domesticated subgroups.

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.

Evolution and diversification of CaM/CML gene family in green plants.

Calmodulin (CaM) and calmodulin-like (CML) proteins are crucial Ca2+ sensors, which are widely involved in different biological processes of plants, including their growth and development, and stress responses. However, the origin and evolution of the CaM/CML gene family in plants remain elusive. In this study, 2133 CaM and 23094 CML genes were identified from the 1000 plants project (1 KP) species and the sequenced plants, covering algae, mosses, monilophytes, lycophytes, flowering plants, and all other green plant branches. Analysis showed that the size of the CML subfamily was correlated with the genome size of corresponding plant species, as well as the total gene number in the genome. Moreover, with the evolution from algae to angiosperms, the number of CML genes in plants increased gradually which could have been driven mainly by genome-wide segmental duplication events, while the number of CaMs remained basically stable at 2-3. Phylogenetic analysis demonstrated that CaM first appeared in green algae, while CML appeared earlier and has already been presented in dinoflagellates. Further analysis showed that the number and sequence of EF-hand domain in CaMs are highly conserved, while those of CMLs are diverse among different plant taxa. Expression analysis revealed that the expression level of CaMs was generally higher than that of CMLs, indicating that the high-expression genes have essential functions, while the low-expression genes are the main reasons for the functional diversity of the CaM/CML gene family in plants. The results might contribute to understanding the evolution of CaM/CML genes as well as their molecular functions.

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.

Proteomic and metabolomic analyses showing the differentially accumulation of NnUFGT2 is involved in the petal red-white bicolor pigmentation in lotus (Nelumbo nucifera).

Bicolor flower lotus is rare with high ornamental value. During the long history of breeding and artificial selection, a very famous lotus cultivar 'Da Sajin' with red and white picotee bicolor petals were obtained. In order to reveal the mechanism underlying the formation of its picotee bicolor pattern in the petal, an integrative metabolomics and proteomics analyses were conducted between red and white parts of its petals. The results showed that the defect of anthocyanidin 3-O-glucosyltransferases (UFGTs) accumulation resulted in the failure of the glycosylation of anthocyanidin, the last step of anthocyanin biosynthesis in white part of the petals. And proteomic data and biochemical analysis showed that the defect of UFGTs accumulation is not related to their transcription, but because of their degradation. Function of one differentially accumulated NnUFGT were proven being involved in anthocyanin biosynthesis through both in-vitro enzyme assay and in-vivo transgenic analyses. This regulation on the protein accumulation of structural genes in anthocyanin biosynthesis was not explored in any other plants, and hence supposed to be a novel mechanism for the formation of picotee bicolor pattern flower. The results not only provide some new insights into the understanding of lotus flower coloration, but also might assist the breeding of flower lotus.

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.

Identifying RNA Modifications by Direct RNA Sequencing Reveals Complexity of Epitranscriptomic Dynamics in Rice.

Transcriptome analysis based on high-throughput sequencing of a cDNA library has been widely applied to functional genomic studies. However, the cDNA dependence of most RNA sequencing techniques constrains their ability to detect base modifications on RNA, which is an important element for the post-transcriptional regulation of gene expression. To comprehensively profile the N6-methyladenosine (m6A) and N5-methylcytosine (m5C) modifications on RNA, direct RNA sequencing (DRS) using the latest Oxford Nanopore Technology was applied to analyze the transcriptome of six tissues in rice. Approximately 94 million reads were generated, with an average length ranging from 619 nt to 1013 nt, and a total of 45,707 transcripts across 34,763 genes were detected. Expression profiles of transcripts at the isoform level were quantified among tissues. Transcriptome-wide mapping of m6A and m5C demonstrated that both modifications exhibited tissue-specific characteristics. The transcripts with m6A modifications tended to be modified by m5C, and the transcripts with modifications presented higher expression levels along with shorter poly(A) tails than transcripts without modifications, suggesting the complexity of gene expression regulation. Gene Ontology analysis demonstrated that m6A- and m5C-modified transcripts were involved in central metabolic pathways related to the life cycle, with modifications on the target genes selected in a tissue-specific manner. Furthermore, most modified sites were located within quantitative trait loci that control important agronomic traits, highlighting the value of cloning functional loci. The results provide new insights into the expression regulation complexity and data resource of the transcriptome and epitranscriptome, improving our understanding of the rice genome.

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.

Integration of metabolomics and transcriptomics analyses investigates the accumulation of secondary metabolites in maturing seed plumule of sacred lotus (Nelumbo nucifera).

Lotus seed plumule (LP) is rich in a variety of antioxidant and anti-inflammatory secondary metabolites, making it a traditional food and medicine widely used in China. Physiological and histological evidences indicated that LP mainly accumulated metabolites in 15-24 days after pollination (DAP) during their development. To systematically investigate the dynamic accumulation of major secondary metabolites, the UPLC-HRMS-based widely targeted metabolomics analyses were performed on maturing LP at 15, 18, 21, and 24 DAP. In total, 767 metabolites were identified, including many secondary metabolites, e.g., 27 % flavonoids and 8 % alkaloids. Among them, 591 were identified as differentially accumulated metabolites (DAMs). The majority of secondary metabolites showed great accumulation after 18 DAP even at the late stage of LP maturation, such as hesperidin, neohesperidin, orobol, serotonin, and lotus special O-nornuciferine, endowing mature LP with effective pharmaceutical properties. The paralleled transcriptomic analysis identified 11,019 differentially expressed genes (DEGs). Based on the comprehensive data, several systematical metabolic regulation maps were established for different secondary metabolites, and 18 DAP was found as a switching point for LP maturing from active primary metabolism to massive secondary metabolites deposition. This study provides valuable information for understanding the mechanism of secondary metabolite accumulation in maturing LP and facilitates its pharmaceutical application.

TMT-based quantitative proteomic and physiological analyses on lotus plumule of artificially aged seed in long-living sacred lotus Nelumbo nucifera.

Seed longevity is important for the maintenance of seed nutritional quality, vigor, and germination potential during storage. Sacred lotus is known as one of the longest living seeds in the world and their ability to maintain longevity has been widely investigated. In this study, a suitable controlled deterioration treatment (CDT) method was first established to evaluate the vigor loss of lotus plumule (LP), and then the Tandem Mass Tags (TMT)-based proteomic analysis was performed on LP from the CDT-treated seed to quantitatively and qualitatively analyze the protein profile dynamic. In total, 4002 proteins were successfully quantified, of them, 558 differently accumulated proteins (DAPs) were identified. Protein processing and RNA-related proteins were found more easily to be affected by CDT, which may directly result in seed vigor loss. Meanwhile, CDT resulted in remarkable up-regulation of numerous proteins related to antioxidation, photosynthesis, RNA and DNA stability, starch and sucrose mobilization, and cell membrane and wall stability, which potentially played key roles in maintaining the lotus seed vigor under CDT. Histological and physiological analyses were also performed to verify some proteome results. This study provided both fundamental data and new insights to further uncover the secret of lotus seed longevity. SIGNIFICANCE: Seed aging affects the seed quality and can result in direct economic losses. The exceptional longevity of sacred lotus seed has attracted extensive attention. In this study, an optimized CDT method was used to mimic the natural aging process of sacred lotus seed, and based on TMT-based quantitative proteomic analysis on the LP profile of CDT-treated seeds, a series of differentially accumulation of specific proteins (DEPs) were revealed related to CDT resistance. Correspondingly, the physiological state and histological structure of the LP along with the CDT were detected to verify the proteome data. This study provided comprehensive information for the molecular basis of lotus seed aging analysis and facilitate to screen seed longevity related proteins for other plant species.

Genome-Wide Identification and Characterization of Heat Shock Protein 20 Genes in Maize.

Maize is an important cereal crop worldwide and is sensitive to abiotic stresses in fluctuant environments that seriously affect its growth, yield, and quality. The small heat shock protein (HSP20) plays a crucial role in protecting plants from abiotic stress. However, little is known about HSP20 in maize (ZmHSP20). In this study, 44 ZmHSP20s were identified, which were unequally distributed over 10 chromosomes, and 6 pairs of ZmHSP20s were tandemly presented. The gene structure of ZmHSP20s was highly conserved, with 95% (42) of the genes having no more than one intron. The analysis of the cis-element in ZmHSP20s promoter demonstrated large amounts of elements related to hormonal and abiotic stress responses, including abscisic acid (ABA), high temperature, and hypoxia. The ZmHSP20s protein had more than two conserved motifs that were predictably localized in the cytoplasm, nucleus, endoplasmic reticulum, peroxisome, mitochondria, and plasma. Phylogenetic analysis using HSP20s in Arabidopsis, rice, maize, and Solanum tuberosum indicated that ZmHSP20s were classified into 11 categories, of which each category had unique subcellular localization. Approximately 80% (35) of ZmHSP20 were upregulated under heat stress at the maize seedling stage, whereas the opposite expression profiling of 10 genes under 37 and 48 °C was detected. A total of 20 genes were randomly selected to investigate their expression under treatments of ABA, gibberellin (GA), ethylene, low temperature, drought, and waterlogging, and the results displayed that more than half of these genes were downregulated while ZmHSP20-3, ZmHSP20-7, ZmHSP20-24, and ZmHSP20-44 were upregulated under 1 h treatment of ethylene. A yeast-one-hybrid experiment was conducted to analyze the binding of four heat stress transcription factors (ZmHSFs) with eight of the ZmHSP20s promoter sequences, in which ZmHSF3, ZmHSF13, and ZmHSF17 can bind to most of these selected ZmHSP20s promoters. Our results provided a valuable resource for studying HSP20s function and offering candidates for genetic improvement under abiotic stress.

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.

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.

Comparative transcriptomic analysis provides insight into carpel petaloidy in lotus (Nelumbo nucifera).

Lotus (Nelumbo nucifera) is a highly recognized flower with high ornamental value. Flower color and flower morphology are two main factors for flower lotus breeding. Petaloidy is a universal phenomenon in lotus flowers. However, the genetic regulation of floral organ petaloidy in lotus remains elusive. In this study, the transcriptomic analysis was performed among three organs, including petal, carpel petaloidy, and carpel in lotus. A total of 1,568 DEGs related to carpel petaloidy were identified. Our study identified one floral homeotic gene encoded by the MADS-box transcription factor, AGAMOUS (AG) as the candidate gene for petaloid in lotus. Meanwhile, a predicted labile boundary in floral organs of N. nucifera was hypothesized. In summary, our results explored the candidate genes related to carpel petaloidy, setting a theoretical basis for the molecular regulation of petaloid phenotype.

Metabolomic Analysis on the Petal of 'Chen Xi' Rose with Light-Induced Color Changes.

Flower color is one of the most prominent traits of rose flowers and determines their ornamental value. The color of the "Chen Xi" rose can change from yellow to red during flower blooming. In the present study, the flavonoid metabolites were investigated by the UPLC-ESI-MS/MS from the petals of four successive flower development stages under natural conditions. In total, 176 flavonoid components, including 49 flavones, 59 flavonols, 12 flavanones, 3 isoflavones, 12 anthocyanins, and 41 proanthocyanidins were identified, with some of them being detected for the first time in this study. Additionally, there were 56 compounds that showed differences among comparison groups, mainly being enriched in pathways of isoflavone, flavonoid, flavone, flavonol, phenylpropanoids, and anthocyanin. Among them, it is anthocyanins that allow the rose flower to turn red when exposed to sunlight. To verify this result, compounds from rose petal with shading treatment (S2D) was also detected but could be clearly separated from the four samples under light by clustering and principal component analyses (PCA). Consistent with low anthocyanins accumulation, the flower with shading could not turn red. Moreover, it provides a foundation for further research on the light-induced color modification of flower.

Protein Phosphorylation Response to Abiotic Stress in Plants.

Plants are an important part of nature because as photoautotrophs, they provide a nutrient source for many other living organisms. Due to their sessile nature, to overcome both biotic and abiotic stresses, plants have developed intricate mechanisms for perception of and reaction to these stresses, both on an external level (perception) and on an internal level (reaction). Specific proteins found within cells play crucial roles in stress mitigation by enhancing cellular processes that facilitate the plants survival during the unfavorable conditions. Well before plants are able to synthesize nascent proteins in response to stress, proteins which already exist in the cell can be subjected to an array of posttranslation modifications (PTMs) that permit a rapid response. These activated proteins can, in turn, aid in further stress responses. Different PTMs have different functions in growth and development of plants. Protein phosphorylation, a reversible form of modification has been well elucidated, and its role in signaling cascades is well documented. In this mini-review, we discuss the integration of protein phosphorylation with other components of abiotic stress-responsive pathways including phytohormones and ion homeostasis. Overall, this review demonstrates the high interconnectivity of the stress response system in plants and how readily plants are able to toggle between various signaling pathways in order to survive harsh conditions. Most notably, fluctuations of the cytosolic calcium levels seem to be a linking component of the various signaling pathways.