The LARGE2-APO1/APO2 regulatory module controls panicle size and grain number in rice.

Panicle size and grain number are important agronomic traits and influence grain yield in rice (Oryza sativa), but the molecular and genetic mechanisms underlying panicle size and grain number control remain largely unknown in crops. Here we report that LARGE2 encodes a HECT-domain E3 ubiquitin ligase OsUPL2 and regulates panicle size and grain number in rice. The loss of function large2 mutants produce large panicles with increased grain number, wide grains and leaves, and thick culms. LARGE2 regulates panicle size and grain number by repressing meristematic activity. LARGE2 is highly expressed in young panicles and grains. Biochemical analyses show that LARGE2 physically associates with ABERRANT PANICLE ORGANIZATION1 (APO1) and APO2, two positive regulators of panicle size and grain number, and modulates their stabilities. Genetic analyses support that LARGE2 functions with APO1 and APO2 in a common pathway to regulate panicle size and grain number. These findings reveal a novel genetic and molecular mechanism of the LARGE2-APO1/APO2 module-mediated control of panicle size and grain number in rice, suggesting that this module is a promising target for improving panicle size and grain number in crops.

Systematic analysis of the ARF gene family in Fagopyrum dibotrys and its potential roles in stress tolerance.

The auxin response factor (ARF) is a plant-specific transcription factor that regulates the expression of auxin response genes by binding directly to their promoters. They play an important role in the regulation of plant growth and development, as well as in the response to biotic and abiotic stresses. However, the identification and functional analysis of ARFs in Fagopyrum dibotrys are still unclear. In this study, a total of 26 FdARF genes were identified using bioinformatic methods. Their chromosomal location, gene structure, physical and chemical properties of their encoded protein, subcellular location, phylogenetic tree, conserved motifs and cis-acting elements in FdARF promoters were analyzed. The results showed that 26 FdARF genes were unevenly distributed on 8 chromosomes, with the largest distribution on chromosome 4 and the least distribution on chromosome 3. Most FdARF proteins are located in the nucleus, except for the proteins FdARF7 and FdARF21 located to the cytoplasm and nucleus, while FdARF14, FdARF16, and FdARF25 proteins are located outside the chloroplast and nucleus. According to phylogenetic analysis, 26 FdARF genes were divided into 6 subgroups. Duplication analysis indicates that the expansion of the FdARF gene family was derived from segmental duplication rather than tandem duplication. The prediction based on cis-elements of the promoter showed that 26 FdARF genes were rich in multiple stress response elements, suggesting that FdARFs may be involved in the response to abiotic stress. Expression profiling analysis showed that most of the FdARF genes were expressed in the roots, stems, leaves, and tubers of F. dibotrys, but their expression exhibits a certain degree of tissue specificity. qRT-PCR analysis revealed that most members of the FdARF gene were up- or down-regulated in response to abiotic stress. The results of this study expand our understanding of the functional role of FdARFs in response to abiotic stress and lay a theoretical foundation for further exploration of other functions of FdARF genes.

The auxin response factor (ARF) gene family in Cyclocarya paliurus: genome-wide identification and their expression profiling under heat and drought stresses.

Auxin response factors (ARFs), as the main components of auxin signaling, play a crucial role in various processes of plant growth and development, as well as in stress response. So far, there have been no reports on the genome-wide identification of the ARF transcription factor family in Cyclocarya paliurus, a deciduous tree plant in the family Juglaceae. In this study, a total of 34 CpARF genes were identified based on whole genome sequence, and they were unevenly distributed on 16 chromosomes, with the highest distribution on chromosome 6. Domain analysis of CpARF proteins displayed that 31 out of 34 CpARF proteins contain a typical B3 domain (DBD domain), except CpARF12/ CpARF14/CpARF31, which all belong to Class VI. And 20 CpARFs (58.8%) contain an auxin_IAA binding domain, and are mainly distributed in classes I, and VI. Phylogenetic analysis showed that CpARF was divided into six classes (I-VI), each containing 4, 4, 1, 8, 4, and 13 members, respectively. Gene duplication analysis showed that there are 14 segmental duplications and zero tandem repeats were identified in the CpARF gene family of the C. paliurus genome. The Ka/Ks ratio of duplicate gene pairs indicates that CpARF genes are subjected to strong purification selection pressure. Synteny analysis showed that C. paliurus shared the highest homology in 74 ARF gene pairs with Juglans regia, followed by 73, 51, 25, and 11 homologous gene pairs with Populus trichocarpa, Juglans cathayensis, Arabidopsis, and rice, respectively. Promoter analysis revealed that 34 CpARF genes had cis-elements related to hormones, stress, light, and growth and development except for CpARF12. The expression profile analysis showed that almost all CpARF genes were differentially expressed in at least one tissue, and several CpARF genes displayed tissue-specific expression. Furthermore, 24 out of the 34 CpARF genes have significantly response to drought stress (P < 0.05), and most of them (16) being significantly down-regulated under moderate drought treatment. Meanwhile, the majority of CpARF genes (28) have significantly response to drought stress (P < 0.05), and most of them (26) are significantly down-regulated under severe drought treatment. Furthermore, 32 out of the 34 CpARF genes have significantly response to high, middle, and low salt stress under salt treatment (P < 0.05). Additionally, subcellular localization analysis confirmed that CpARF16 and CpARF32 were all localized to nucleus. Thus, our findings expand the understanding of the function of CpARF genes and provide a basis for further functional studies on CpARF genes in C. paliurus. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01474-1.

Genome-wide analysis of the HSP20 gene family and its response to heat and drought stress in Coix (Coix lacryma-jobi L.).

BACKGROUND: Heat shock protein 20 (HSP20) is a member of the heat stress-related protein family, which plays critical roles in plant growth, development, and response to abiotic stresses. Although many HSP20 genes have been associated with heat stress in numerous types of plants, little is known about the details of the HSP20 gene family in Coix. To investigate the mechanisms of the ClHSP20 response to heat and drought stresses, the ClHSP20 gene family in Coix was identified and characterized based on genome-wide analysis. RESULTS: A total of 32 putative ClHSP20 genes were identified and characterized in Coix. Phylogenetic analysis indicated that ClHSP20s were grouped into 11 subfamilies. The duplicated event analysis demonstrated that tandem duplication and segment duplication events played crucial roles in promoting the expansion of the ClHSP20 gene family. Synteny analysis showed that Coix shared the highest homology in 36 HSP20 gene pairs with wheat, followed by 22, 19, 15, and 15 homologous gene pairs with maize, sorghum, barley, and rice, respectively. The expression profile analysis showed that almost all ClHSP20 genes had different expression levels in at least one tissue. Furthermore, 22 of the 32 ClHSP20 genes responded to heat stress, with 11 ClHSP20 genes being significantly upregulated and 11 ClHSP20 genes being significantly downregulated. Furthermore, 13 of the 32 ClHSP20 genes responded to drought stress, with 6 ClHSP20 genes being significantly upregulated and 5 ClHSP20 genes being significantly downregulated. CONCLUSIONS: Thirty-two ClHSP20 genes were identified and characterized in the genome of Coix. Tandem and segmental duplication were identified as having caused the expansion of the ClHSP20 gene family. The expression patterns of the ClHSP20 genes suggested that they play a critical role in growth, development, and response to heat and drought stress. The current study provides a theoretical basis for further research on ClHSP20s and will facilitate the functional characterization of ClHSP20 genes.

Assembly and phylogenetic analysis of the mitochondrial genome of endangered medicinal plant Huperzia crispata.

Huperzia crispata is a traditional Chinese herb plant and has attracted special attention in recent years for its products Hup A can serve as an acetylcholinesterase inhibitor (AChEI). Although the chloroplast (cp) genome of H. crispata has been studied, there are no reports regarding the Huperzia mitochondrial (mt) genome since the previously reported H. squarrosa has been revised as Phlegmariurus squarrosus. The mt genome of H. crispata was sequenced using a combination of long-read nanopore and Illumina sequencing platforms. The entire H. crispata mt genome was assembled in a circular with a length of 412,594 bp and a total of 91 genes, including 45 tRNAs, 6 rRNAs, 37 protein-coding genes (PCGs), and 3 pseudogenes. Notably, the rps8 gene was present in P. squarrosus and a pseudogene rps8 was presented in H. crispata, which was lacking in most of Pteridophyta and Gymnospermae. Intron-encoded maturase (mat-atp9i85 and mat-cobi787) genes were present in H. crispata and P. squarrosus, but lost in other examined lycophytes, ferns, and Gymnospermae plants. Collinearity analysis showed that the mt genome of H. crispata and P. squarrossus is highly conservative compared to other ferns. Relative synonymous codon usage (RSCU) analysis showed that the amino acids most frequently found were phenylalanine (Phe) (4.77%), isoleucine (Ile) (4.71%), lysine (Lys) (4.26%), while arginine (Arg) (0.32%), and histidine (His) (0.42%) were rarely found. Simple sequence repeats (SSR) analysis revealed that a total of 114 SSRs were identified in the mt genome of H. crispata and account for 0.35% of the whole mt genome. Monomer repeats were the majority types of SSRs and represent 91.89% of the total SSRs. In addition, a total of 1948 interspersed repeats (158 forward, 147 palindromic, and 5 reverse repeats) with a length ranging from 30 bp to 14,945 bp were identified in the H. crispata mt genome and the 30-39-bp repeats were the most abundant type. Gene transfer analysis indicated that a total of 12 homologous fragments were discovered between the cp and mt genomes of H. crispata, accounting for 0.93% and 2.48% of the total cp and mt genomes, respectively. The phylogenetic trees revealed that H. crispata was the sister of P. squarrosus. The Ka/Ks analysis results suggested that most PCGs, except atp6 gene, were subject to purification selection during evolution. Our study provides extensive information on the features of the H. crispata mt genome and will help unravel evolutionary relationships, and molecular identification within lycophytes.

Control of Grain Size and Weight by the GSK2-LARGE1/OML4 Pathway in Rice.

Regulation of grain size is crucial for improving crop yield and is also a basic aspect in developmental biology. However, the genetic and molecular mechanisms underlying grain size control in crops remain largely unknown despite their central importance. Here, we report that the MEI2-LIKE PROTEIN4 (OML4) encoded by the LARGE1 gene is phosphorylated by GLYCOGEN SYNTHASE KINASE2 (GSK2) and negatively controls grain size and weight in rice (Oryza sativa). Loss of function of OML4 leads to large and heavy grains, while overexpression of OML4 causes small and light grains. OML4 regulates grain size by restricting cell expansion in the spikelet hull. OML4 is expressed in developing panicles and grains, and the GFP-OML4 fusion protein is localized in the nuclei. Biochemical analyses show that the GSK2 physically interacts with OML4 and phosphorylates it, thereby possibly influencing the stability of OML4. Genetic analyses support that GSK2 and OML4 act, at least in part, in a common pathway to control grain size in rice. These results reveal the genetic and molecular mechanism of a GSK2-OML4 regulatory module in grain size control, suggesting that this pathway is a suitable target for improving seed size and weight in crops.

Diversity of endophytic fungal community in Huperzia serrata from different ecological areas and their correlation with Hup A content.

BACKGROUND: Huperzine A (Hup A) has attracted considerable attention as an effective therapeutic candidate drug used to treat Alzheimer's disease. Whereas, the production of Hup A from wild plants faced a major challenge, which is the wild Huperzia Serrata harbor a low Hup A content, has a long-life cycle, and has a small yield. At present, several reports showed that Hup A is produced by various endophytic fungal strains isolated from H. serrata, thereby providing an alternative method to produce the compound and reduce the consumption of this rare and endangered plant. However, till now, very few comprehensive studies are available on the biological diversity and structural composition of endophytic fungi and the effects of endophytic fungi on the Hup A accumulation in H. serrata. RESULTS: In this research, the composition and diversity of fungal communities in H. serrata were deciphered based on high-throughput sequencing technology of fungal internal transcribed spacer regions2 (ITS2). The correlation between endophytic fungal community and Hup A content was also investigated. Results revealed that the richness and the diversity of endophytic fungi in H. serrata was various according to different tissues and different ecological areas. The endophytic fungal communities of H. serrata exhibit species-specific, ecological-specific, and tissue-specific characteristics. There are 6 genera (Ascomycota_unclassified, Cyphellophora, Fungi_unclassified, Sporobolomyces, and Trichomeriaceae_unclassified) were significantly positively correlated with Hup A content in all two areas, whereas, there are 6 genera (Auricularia, Cladophialophora, Cryptococcus, Mortierella, and Mycena) were significantly negatively correlated with Hup A content of in all two areas. CONCLUSIONS: This study indicated a different composition and diverse endophytic fungal communities in H. serrata from different organs and ecological areas. The current study will provide the realistic basis and theoretical significance for understanding the biological diversity and structural composition of endophytic fungal communities in H. serrata, as well as providing novel insights into the interaction between endophytic fungi and Hup A content.

Structure characterization and bioactivity of neutral polysaccharides from different sources of Polygonatum Mill.

Polygonati rhizoma (PR), a traditional medical and edible product, is rich in polysaccharides and exhibits physiological activity, including antioxidant, hypoglycemic and hypolipidemic properties. Neutral polysaccharides have been reported to be one of the main active ingredients of Polygonatum, with many of these fractions being responsible for the biological activity. This behavior was shown to be closely connected to the chemical structure, monosaccharide composition, and glycosidic bond type. There are few reports on the chemical constituents of the neutral polysaccharides from different sources of PR. In this study, neutral polysaccharides of PR from four different regions of China (Chun'an (Zhejiang), Xixia (Henan), Danfeng (Shanxi), and Pan'an (Zhejiang)), named CAZJ, XXHN, DFSX, and PAZJ, respectively, were isolated by anion-exchange and gel-permeation chromatography. Structures of the four polysaccharides were investigated. The results showed that all of them were mainly glucose and mannose, while the monosaccharide composition and content of polysaccharides from different sources varied. The molecular weights of CAZJ, XXHN, DFSX, and PAZJ were 14.119, 22.352, 18.127, and 15.699 kDa, respectively. Infrared spectra illustrated the existence of α-glycosidic bond and ß-glycosidic bond in the polysaccharides. CAZJ, XXHN, and DFSX possessed a pyranose ring structure, whereas PAZJ had a furanose ring structure. Congo red test indicated that XXHN, DFSX, and PAZJ had a triple-helix structure. X-ray diffraction showed that the polysaccharides consisted of crystalline and amorphous regions. All four polysaccharides exhibited different degrees of antioxidant and hypoglycemic activities with a dose-dependent manner in the 1.0-10.0 mg/mL concentration range. Correlation analysis revealed that the bioactivities of polysaccharides was significantly related to monosaccharide composition, uronic acid, and protein content. The results suggested that neutral polysaccharides could be used as potential natural antioxidants and hypoglycemic agents for functional and nutraceutical applications.

The complete chloroplast genome of the medical plant Huperzia crispata from the Huperziaceae family: structure, comparative analysis, and phylogenetic relationships.

BACKGROUND: Huperzia crispata, belonging to the Huperziaceae family, is one of the most essential resources of huperzine A for candidate drugs to treat Alzheimer's diseases. However, there is very limited information about H. crispat, and its taxonomic status and interspecific relationships between Huperzia species are still unclear. To investigate the taxonomic classification of Huperzia species and identify species discrimination markers, the complete chloroplast (cp) genome of H. crispata was sequenced and characterized for the first time. METHODS AND RESULTS: Total genomic DNA was isolated and sequenced using the next-generation Illumina NovaSeq 6000 platform. The data were filtered, assembled and annotated by a series software and web service. The results were as follows: the cp genome of H. crispata was 154,320 bp long with a large single-copy (LSC) region of 104,023 bp, a small single-copy (SSC) region of 19,671 bp, and a pair of inverted repeat (IRa and IRb) regions of 15,313 bp. A total of 131 genes, including 87 protein-coding genes, 36 transfer RNA genes (tRNAs), and eight ribosome RNA genes (rRNAs), were annotated in the cp genome. The contraction and expansion of the inverted repeat (IR) regions were relatively conserved in the Huperzia genus. Codon usage bias analysis showed that the encoding rate at the 3-end of codon A/T (74.34%) was significantly higher than that of C/G (25.66%). A total of 8 hotspot loci with high Pi values (> 0.06) were identified in the four Huperzia species based on nucleic acid diversity analysis. Ka/Ks selective pressure analysis demonstrated that the cemA gene is the most common gene undergoing positive selection among Huperzia. In addition, a total of 261 simple sequence repeats and 179 interspersed repeats were identified in the cp genome. Phylogenetic tree analysis based on the complete protein sequences of 23 related species of H. crispata indicated that H. serrata f. longipetiolata is a sister of H. crispata, suggesting that H. serrata f. longipetiolata and H. crispata are more closely related than H. serrata and H. lucidula. CONCLUSIONS: The results strongly supported that H. crispata was more closely related to H. serrata f. longipetiolata than to H. serrata and H. lucidula within the Huperzia genus. The outcome provided important information for the phylogenetic analysis of the subsequent specific molecular species identification in Huperzia. The present results will provide valuable information for further research into the classification, phylogeny and species identification of Huperzia plants.

Transcriptome Analysis of Different Sections of Rhizome in Polygonatum sibiricum Red. and Mining Putative Genes Participate in Polysaccharide Biosynthesis.

As a traditional Chinese herb, the rhizomes of Polygonatum sibiricum Red. are rich in various compounds which have plenty of pharmacological applications and biological activities. Among them, Polygonatum sibiricum polysaccharides (PSP) are the main active ingredients and exhibit a broad range of pharmacological. Based on previous researches, identifying genes involved in PSP biosynthesis will help delineate such pathway at the molecular level. In that case, we performed RNA sequencing analysis for two sections of P. sibiricum Red.'s rhizomes significantly different in PSP content. A total of 435,858 unigenes were obtained by assembling transcripts from both sections and 29,548 (6.77%) ones were annotated in all seven public databases. Analyzing count data of RNA-seq, 13,460 differential expression genes (DEGs) between two sections of rhizomes were acquired. After DEGs were mapped to KEGG databases, twelve represented KEGG pathways related to PSP biosynthesis were summed up. And most DEGs were assigned to the pathway of "Starch and sucrose metabolism". Finally, seventeen candidate genes whose expression levels were related to the polysaccharide content, were considered involving PSP biosynthesis in P. sibiricum Red. The present study lays a foundation of researching the molecular mechanisms of PSP biosynthesis.