蔡泽坪课题组

（海南大学热带农林学院）

1. Cai ZP, Dai YN, Jin X, Xu H, Huang Z, Xie ZY, Wang XC, Yu XD and Luo JJ. Ambient temperature regulates root circumnutation in rice through the ethylene pathway: transcriptome analysis reveals key genes involved[J]. Front. Plant Sci., 2024, 15:1348295.

https://doi.org/10.3389/fpls.2024.1348295

Plant roots are constantly prepared to adjust their growth trajectories to avoid unfavorable environments, and their ability to reorient is particularly crucial for survival. Under laboratory conditions, this continuous reorientation of the root tip is manifested as coiling or waving, which we refer to as root circumnutation. However, the effect of ambient temperature (AT) on root circumnutation remains unexplored. In this study, rice seedlings were employed to assess the impact of varying ATs on root circumnutation. The role of ethylene in mediating root circumnutation under elevated AT was examined using the ethylene biosynthesis inhibitor aminooxyacetic acid (AOA) and the ethylene perception antagonist silver thiosulfate (STS). Furthermore, transcriptome sequencing, weighted gene co-expression network analysis, and real-time quantitative PCR were utilized to analyze gene expressions in rice root tips under four distinct treatments: 25°C, 35°C, 35°C+STS, and 35°C+AOA. As a result, genes associated with ethylene synthesis and signaling (OsACOs and OsERFs), auxin synthesis and transport (OsYUCCA6, OsABCB15, and OsNPFs), cell elongation (OsEXPAs, OsXTHs, OsEGL1, and OsEXORDIUMs), as well as the inhibition of root curling (OsRMC) were identified. Notably, the expression levels of these genes increased with rising temperatures above 25°C. This study is the first to demonstrate that elevated AT can induce root circumnutation in rice via the ethylene pathway and proposes a potential molecular model through the identification of key genes. These findings offer valuable insights into the growth regulation mechanism of plant roots under elevated AT conditions.

2. Xiao X, Wu FH, Wang B, Cai ZP, Wang LY, Zhang YF, Yu XD, Luo YP. Clerodendranthus spicatus (Thunb.) Water Extracts Reduce Lipid Accumulation and Oxidative Stress in the Caenorhabditis elegans. Int J Mol Sci. 2024 Sep 6;25(17):9655.

https://doi.org/10.3390/ijms25179655‍

Clerodendranthus spicatus (Thunb.) (Kidney tea) is a very distinctive ethnic herbal medicine in China. Its leaves are widely used as a healthy tea. Many previous studies have demonstrated its various longevity-promoting effects; however, the safety and specific health-promoting effects of Clerodendranthus spicatus (C. spicatus) as a dietary supplement remain unclear. In order to understand the effect of C. spicatus on the longevity of Caenorhabditis elegans (C. elegans), we evaluated its role in C. elegans; C. spicatus water extracts (CSw) were analyzed for the major components and the effects on C. elegans were investigated from physiological and biochemical to molecular levels; CSw contain significant phenolic components (primarily rosmarinic acid and eugenolinic acid) and flavonoids (primarily quercetin and isorhamnetin) and can increase the lifespan of C. elegans. Further investigations showed that CSw modulate stress resistance and lipid metabolism through influencing DAF-16/FoxO (DAF-16), Heat shock factor 1 (HSF-1), and Nuclear Hormone Receptor-49 (NHR-49) signalling pathways; CSw can improve the antioxidant and hypolipidemic activity of C. elegans and prolong the lifespan of C. elegans (with the best effect at low concentrations). Therefore, the recommended daily use of C. spicatus should be considered when consuming it as a healthy tea on a daily basis.

3. Xu ST, Wang YC, Yu XD, Cai ZP and Ren MX (2024) Suspended soils enrich local forest floor soils during the rainy season in a tropical monsoon rainforest of Hainan Island, South China. Front. Plant Sci. 15:1415754.

https://doi.org/10.3389/fpls.2024.1415754

Epiphytic plants are abundant in rainforests and often serve as traps for litter and dust falling from the canopy. As it accumulates, this material can form nutrient rich soils, which are likely involved in local nutrient cycling and ecological processes. Toexplorespatial andtemporalvariation in the influenceof suspended soils on local nutrient cycles, we compared the physical, chemical and biological properties of suspended soils from the locally-dominant epiphytic Bird’s nest fern (Asplenium nidus L.) to those of three types offorest floor soils (soil collected from upslope, downslope, and underneath the host tree) in a tropical monsoon rainforest in Bawangling National Nature Reserve on Hainan Island, China. Suspended and forest floor soils were all acidic, with suspended soils having much higher organic matter (66.84%) and water content (~ 300%) than forest floor soils. Suspended soils contained significantly more available nitrogen, phosphorous, and potassium and had much higher urease, cellulase, and catalase activities, indicating that they harbored diverse microbial communities with higher decomposition and biomineralization activity. Physicochemicaltraits ofsuspendedsoilandsoilcollectedfromunder the host tree were significantly more similar in the rainy season than in the dry season, suggesting that suspended soils may contribute to local nutrient cycling as they are flushed out of epiphytic plants and enrich stemflow and forest floor soils. Thus, suspended soils play a role in local nutrient cycling, especially during the rainy season. This study provides empirical support for the seasonality and heterogeneity of forest floor soil enrichment by suspended soils in tropical monsoon rainforests.

Higher AT upregulates the expression of a series of genes by promoting the biosynthesis andsignaling of ethylene. These include genes involved in the synthesis and transport of auxin (such as OsYUCCA6, OsABCB15, and OsNPFs), as well as genes involved in cell elongation (such as OsEXPAs, OsXTHs, OsEGL1, and OsEXORDIUMs), ultimately leading to root circumnutation. Meanwhile, OsRMC plays a negative regulatory role in this process.

Effect of CSw with different concentrations on the life span of C. elegans. (A) Survival curves of C. elegans treated with different concentrations of CSw (0, 20, 50, 100, 500, and 1000 μg/mL); (B) qualitative observation of fluorescence intensity of lipofuscin; (C) representative pictures of lipofuscin. Different letters (a, b) represent significant (p < 0.05) differences among different groups; the same letters indicate no significant differences between the two groups. The same subscript number (a1, b1, c1) indicates that the significant differences analysis is performed within the same group.

Redundancy analysis results revealed that the main environmental factors affecting soil nutrient status and microbial activity were pH, water content, and temperature in the dry season (A). In the rainy season, organic matter content, moisture content, and available P had the strongest influence (B).

1. Luo JJ, Chen ZJ, Huang R, Wu YH, Liu C, Cai ZP, Dong RS, Arango J, Rao IM, Schultze-Kraft R, Liu GD. and Liu PD. Multi-omics analysis reveals the roles of purple acid phosphatases in organic phosphorus utilization by the tropical legume Stylosanthes guianensis[J]. Plant Journal.. 2023.

https://doi.org/10.1111/tpj.16526

Stylo (Stylosanthes guianensis) is a tropical legume known for its exceptional tolerance to low phosphate(Pi), a trait believed to be linked to its high acid phosphatase (APase) activity. Previous studies haveobserved genotypic variations in APase activity in stylo; however, the gene encoding the crucial APaseresponsible for this variation remains unidentified. In this study, transcriptomic and proteomic analyseswere employed to identify eight Pi starvation-inducible (PSI) APases belonging to the purple APase (PAP)family in the roots of stylo and seven in the leaves. Among these PSI-PAPs, SgPAP7 exhibited a significantlypositive correlation in its expression levels with the activities of both internal APase and root-associated APase across 20 stylo genotypes under low-Pi conditions. Furthermore, the recombinant SgPAP7 displayedhigh catalytic activity toward adenosine 50-diphosphate (ADP) and phosphoenolpyruvate (PEP) in vitro.Overexpression (OE) of SgPAP7 in Arabidopsis facilitated exogenous organic phosphorus utilization. More-over, SgPAP7 OE lines showed lower shoot ADP and PEP levels than the wild type, implying that SgPAP7 isinvolved in the catabolism and recycling of endogenous ADP and PEP, which could be beneficial for plantgrowth in low-Pi soils. In conclusion, SgPAP7 is a key gene with a major role in stylo adaptation to low-Piconditions by facilitating the utilization of both exogenous and endogenous organic phosphorus sources. Itmay also function as a PEP phosphatase involved in a glycolytic bypass pathway that minimizes the needfor adenylates and Pi. Thus, SgPAP7 could be a promising target for improving tolerance of crops to low-Pi availability.

2. Cai ZP, Wang XC, Xie ZY, Wen ZY, Yu XD, Xu ST, Su XY and Luo JJ. Light response of gametophyte in Adiantum flabellulatum: transcriptome analysis and identification of key genes and pathways[J]. Front. Plant Sci., 2023, 14: 1222414.

https://doi.org/10.3389/fpls.2023.1222414

Light serves not only as a signaling cue perceived by plant photoreceptors but also as an essential energy source captured by chloroplasts. However, excessive light can impose stress on plants. Fern gametophytes possess the unique ability to survive independently and play a critical role in the alternation of generations. Due to their predominantly shaded distribution under canopies, light availability becomes a limiting factor for gametophyte survival, making it imperative to investigate their response to light. Previous research on fern gametophytes’ light response has been limited to the physiological level. In this study, we examined the light response of Adiantum flabellulatum gametophytes under different photosynthetic photon flux density (PPFD) levels and identified their high sensitivity to low light. We thereby determined optimal and stress-inducing light conditions. By employing transcriptome sequencing, weighted gene co-expression network analysis, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, we identified 10,995 differentially expressed genes (DEGs). Notably, 3 PHYBs and 5 Type 1 CRYs (CRY1s) were significantly down-regulated at low PPFD (0.1 μmol m^-2 s^-1). Furthermore, we annotated 927 DEGs to pathways related to photosynthesis and 210 to the flavonoid biosynthesis pathway involved in photoprotection. Additionally, we predicted 34 transcription factor families and identified a close correlation between mTERFs and photosynthesis, as well as a strong co-expression relationship between MYBs and bHLHs and genes encoding flavonoid synthesis enzymes. This comprehensive analysis enhances our understanding of the light response of fern gametophytes and provides novel insights into the mechanisms governing their responses to light.

4. Li MZ, Lv MH, Wang XJ, Cai ZP, Yao HR, Zhang DY, Li HQ, Zhu MS, Du WB, Wang RS, Wang Z, Kui H, Hou SW, Li J, Yi J, Gou XP. The EPFL-ERf-SERK signaling controls integument development in Arabidopsis[J]. New Phytol., 2023 Apr;238(1):186-201.

https://doi.org/10.1111/nph.18701‍

As the seed precursor, the ovule produces the female gametophyte (or embryo sac), and the subsequent double fertilization occurs in it. The integuments emerge sequentially from the integument primordia at the early stages of ovule development and finally enwrap the embryo sac gradually during gametogenesis, protecting and nursing the embryo sac. However, the mechanisms regulating integument development are still obscure. In this study, we show that SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES (SERKs) play essential roles during integument development in Arabidopsis thaliana. The serk1/2/3 triple mutant shows arrested integuments and abnormal embryo sacs, similar defects also found in the triple loss-of-function mutants of ERECTA family (ERf) genes. Ovules of serk1/2/3 er erl1/2 show defects similar to er erl1/2 and serk1/2/3. Results of yeast two-hybrid analyses, bimolecular fluorescence complementation (BiFC) analyses, and co-immunoprecipitation assays demonstrated that SERKs interact with ERf, which depends on EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family small peptides. The sextuple mutant epfl1/2/3/4/5/6 shows integument defects similar to both of er erl1/2 and serk1/2/3. Our results demonstrate that ERf–SERK-mediated EPFL signaling orchestrates the development of the female gametophyte and the surrounding sporophytic integuments.

5. Liang JW, Yang J, Yev ZH, Cai ZP, Xu ST, Wang P, and Yu XD . Response of Asplenium nidus to Drought Stress and Roles of AVP1 Genes[J]. American Fern Journal , 2023, 113(1): 14-27.

https://doi.org/10.1640/0002-8444-113.1.14

To explore how Asplenium nidus responds to drought stress and to find V-PPase (AVP1) homologous genes of this species, we conducted greenhouse experiments. We measured a series of physiological and biochemical indices after drought treatment and analyzed the expression of AVP1 homologous genes in A. nidus by RT-qPCR. The results indicated that A. nidus can adapt to drought by limiting stomatal conductance, thus inhibiting plant photosynthesis and reducing physiological activities. In addition, A. nidus can also change its osmotic potential by increasing proline content to maintain normal metabolic activities, and prevent the destruction of reactive oxygen species by increasing the activities of superoxide dismutase (SOD) and peroxidase (POD). According to analysis of the relative expression levels of genes, AVP1-2 and AVP1-4 may be the drought-resistant genes of A. nidus. This study lays a foundation for further exploration of the drought tolerance mechanism and drought-resistant genes of A. nidus.

3. Cai ZP, Zhao XK, Zhou C, Fang T, Liu GD, Luo JJ. Genome Wide Mining of the Tandem Duplicated Type III Polyketide Synthases and Their Expression, Structure Analysis of Senna tora[J]. Int. J. Mol. Sci., 2023, 24, 4837. ‍

https://doi.org/10.3390/ijms24054837‍

Senna tora is one of the homologous crops used as a medicinal food containing an abundance of anthraquinones. Type III polyketide synthases (PKSs) are key enzymes that catalyze polyketide formation; in particular, the chalcone synthase-like (CHS-L) genes are involved in anthraquinone production. Tandem duplication is a fundamental mechanism for gene family expansion. However, the analysis of the tandem duplicated genes (TDGs) and the identification and characterization of PKSs have not been reported for S. tora. Herein, we identified 3087 TDGs in the S. tora genome; the synonymous substitution rates (Ks) analysis indicated that the TDGs had recently undergone duplication. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the type III PKSs were the most enriched TDGs involved in the biosynthesis of the secondary metabolite pathways, as evidenced by 14 tandem duplicated CHS-L genes. Subsequently, we identified 30 type III PKSs with complete sequences in the S. tora genome. Based on the phylogenetic analysis, the type III PKSs were classified into three groups. The protein conserved motifs and key active residues showed similar patterns in the same group. The transcriptome analysis showed that the chalcone synthase (CHS) genes were more highly expressed in the leaves than in the seeds in S. tora. The transcriptome and qRT-PCR analysis showed that the CHS-L genes had a higher expression in the seeds than in other tissues, particularly seven tandem duplicated CHS-L2/3/5/6/9/10/13 genes. The key active-site residues and three-dimensional models of the CHS-L2/3/5/6/9/10/13 proteins showed slight variation. These results indicated that the rich anthraquinones in S. tora seeds might be ascribed to the PKSs’ expansion from tandem duplication, and the seven key CHS-L2/3/5/6/9/10/13 genes provide candidate genes for further research. Our study provides an important basis for further research on the regulation of anthraquinones’ biosynthesis in S. tora.

Relationships between acid phosphatase (APase) activities and expression levels of SgPAP genes among 20 stylo genotypes under low phosphate (Pi) conditions.(a) Correlation analysis of root-internal APase activities and relative expression levels of eight SgPAP genes in the roots. Root-internal APase activity was assessed using adenosine 50-triphosphate (ATP), adenosine 50-diphosphate (ADP), and adenosine 50-monophosphate (AMP), glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), fructose-6-phosphate (F6P), phosphoenolpyruvate (PEP), and 3-phosphoglycerate (3-PGA) as substrates, respectively. (b, c) Correlation analysis of leaf-internal APase activity and relative expression levels of SgPAP7 in the leaves. ADP (b) and PEP (c) were employed as substrates to assess leaf-internal APase activity, respectively. (d) Correlation analysis of root-associated APase activity (using ADP as a substrate) and relative expression levels of SgPAP7 in the roots. 10-day-old seedlings of 20 stylo genotypes were subjected to low-Pi (5 lM KH2PO4)treatment for 18 days. Afterward, leaf and root samples were used to assess APase activity, andthe expression levels of the SgPAP genes were analyzed by qRT-PCR analysis. The Pearson correlation analysis was conducted using the R platform (version 4.0.2).

Co-expression network of flavonoid biosynthesis genes and MYB/bHLH transcription factor genes. (A–D):Co-expression networks of flavonoid biosynthesis genes and MYB/bHLH transcription factor genes in green, brown, yellow, and turquoise modules, respectively. Nodes represent genes, with larger nodes indicating higher degree values. Lines represent co-expression weights between genes, with thicker and darker lines indicating higher weight values.

The genome-wide identification and analysis of the type III PKS superfamily genes. (a) The phylogenetic relationship and the number of type III PKS superfamily genes in Senna tora and other plant species in the Papilionoideae and Caesalpinoideae clades. (b) The distribution, structure, and domain of the type III PKS genes in the S. tora genome. The left side shows the linkage group or scaffold in which the gene is located, the gene ID and structure of type III PKS genes are displayed in the middle location, and the domains predicted by Pfam are given on the left side. (c) The unrooted phylogenetic tree shows the evolutionary relationships of the type III PKS protein sequences from S. tora, Glycine max, Arabidopsis thaliana, and Medicago truncatula. The gene IDs with red represent the TDGs in the S. tora genome.

Ovule defects of serk1/2/3 are caused by loss-of-function of SERK1, SERK2, and SERK3. (a–j) Ovules of wild-type (WT; Col-0) (a–e) and serk1/2/3 (f–j) observed by a differential interference contrast (DIC) microscope. Ovules at stages 2-I (a, f), 2-III (b, g), 3-I (c, h), 3-II/III (d, i), and 3-V (e, j) are shown. Putative development stages for serk1/2/3 are shown (h–j). The nucleus of the megaspore mother cell (MMC) (a, b, f, g) and embryo sac (c, d, e, h, i) is highlighted in red. The MMC (a, b, f, g) and embryo sac (c, d, e, h, i) are outlined by white dots. (k–p) Mature ovules of Col-0 (k), serk1/2/3 (l), serk1/2/3-2 (m), and complemented lines (n–p). A mature ovule of Col-0 (k, stage 3-VI) contains a big vacuole and a big central cell nucleus in the center, three antipodal cells in the chalaza end (one of them is not in the focal plane), an egg cell, and two synergid cells (one of them is not in the focal plane) in the micropyle end. Mature ovules of serk1/2/3 (l) and serk1/2/3-2 (m) show arrested integuments and a small embryo sac without nucleus. Mature ovules of serk1/2/3 complemented by genomic fragments of SERK1 (n), SERK2 (o), and SERK3 (p) show normal morphology similar to the WT. (q–t) Expression patterns of INO in serk1/+/2/3 (q, r) and serk1/2/3 (s, t) ovules were visualized by NLS-YFP driven by an INO promoter (pINO). Merged YFP and DIC figures are shown and embryo sacs are outlined by white dots. The ovules at early stages are outlined by yellow dots. AN, antipodal cell nucleus; CN, central cell nucleus; EN, egg cell nucleus; f, funiculus; ii, inner integument; oi, outer integument; SN, synergid cell nucleus; V, vacuole. Bars, 25 μm.

level of the AnAVP1-1 gene increased on the 2^th week of T1, decreased on the 4^th,and increased after rehydration. During T2 treatment, the gene expression level increased significantly on the 2^nd and 4^th weeks, and decreased significantly on the 6^th and 8^th weeks. The expression level of the AnAVP1-2 gene increased significantly on the 6^th week of T1 treatment and decreased after rehydration. The gene expression level increased significantly on the 2^nd and 4^th weeks of T2 treatment. The expression level of the AnAVP1-3 gene increased significantly on the 4^th week of T1 treatment. The gene expression level increased slightly on the 2^tn and 4^th weeks of T2 treatment. The relative expression level of AnAVP1-4 increased on the 2^nd and 4^th weeks of T1 treatment and decreased after the 6^th week. Under T2, the gene expression level increased significantly on the 4^th week, decreased on the 6^th week and increased again on the 8^th week (Fig. 9; Table S3).

1. Luo JJ, Cai ZP, Huang R, Wu YH, Liu C, Huang CQ, Liu PD, Liu GD and Dong RS. Integrated multi-omics reveals the molecular mechanisms underlying efficient phosphorus use under phosphate deficiency in elephant grass (Pennisetum purpureum)[J]. Front. Plant Sci., 2022, 13: 1069191.

https://doi.org/10.3389/fpls.2022.1069191

Phosphorus (P) is an essential macronutrient element for plant growth, and deficiency of inorganic phosphate (Pi) limits plant growth and yield. Elephant grass (Pennisetum purpureum) is an important fodder crop cultivated widely in tropical and subtropical areas throughout the world. However, the mechanisms underlying efficient P use in elephant grass under Pi deficiency remain poorly understood. In this study, the physiological and molecular responses of elephant grass leaves and roots to Pi deficiency were investigated. The results showed that dry weight, total P concentration, and P content decreased in Pi-deprived plants, but that acid phosphatase activity and P utilization efficiency (PUE) were higher than in Pi-sufficient plants. Regarding Pi starvation-responsive (PSR) genes, transcriptomics showed that 59 unigenes involved in Pi acquisition and transport (especially 18 purple acid phosphatase and 27 phosphate transporter 1 unigenes) and 51 phospholipase unigenes involved in phospholipids degradation or Pi-free lipids biosynthesis, as well as 47 core unigenes involved in the synthesis of phenylpropanoids and flavonoids, were significantly up-regulated by Pi deprivation in leaves or roots. Furthermore, 43 unigenes related to Pi-independent- or inorganic pyrophosphate (PPi)-dependent bypass reactions were markedly up-regulated in Pi-deficient leaves, especially five UDP-glucose pyrophosphorylase and 15 phosphoenolpyruvate carboxylase unigenes. Consistent with PSR unigene expression changes, metabolomics revealed that Pi deficiency significantly increased metabolites of Pi-free lipids, phenylpropanoids, and flavonoids in leaves and roots, but decreased phospholipid metabolites. This study reveals the mechanisms underlying the responses to Pi starvation in elephant grass leaves and roots, which provides candidate unigenes involved in efficient P use and theoretical references for the development of P-efficient elephant grass varieties.

2. Cai ZP, Xie ZY, Wang XC, Zhang SX, Wu Q, Yu XD, Guo Y, Gao SY, Zhang YG, Xu ST, Wang HG, Luo JJ. Excavation of Genes Responsive to Brassinosteroids by Transcriptome Sequencing in Adiantum flabellulatum Gametophytes[J]. Genes, 2022, 13, 1061.

https://doi.org/10.3390/genes13061061

Brassinosteroids (BRs) are a class of polyhydroxysteroid plant hormones; they play important roles in the development and stress resistance of plants. The research on BRs has mainly been carried out in angiosperms, but in ferns—research is still limited to the physiological level and is not in-depth. In this study, Adiantum flabellulatum gametophytes were used as materials and treated with 10−6 M brassinolide (BL). The differentially expressed genes (DEGs) responsive to BRs were identified by transcriptome sequencing, GO, KEGG analysis, as well as a quantitative real-time polymerase chain reaction. From this, a total of 8394 DEGs were screened. We found that the expressions of photosynthetic genes were widely inhibited by high concentrations of BL in A. flabellulatum gametophytes. Moreover, we detected many BR synthase genes, except BR6ox2, which may be why castasterone (CS) rather than BL was detected in ferns. Additionally, we identified (for the first time) that the expressions of BR synthase genes (CYP90B1, CYP90C1, CYP90D1, CPD, and BR6ox1) were negatively regulated by BL in fern gametophytes, which indicated that ferns, including gametophytes, also needed the regulatory mechanism for maintaining BR homeostasis. Based on transcriptome sequencing, this study can provide a large number of gene expression data for BRs regulating the development of fern gametophytes.

4. Ou Y, Tao BQ, Wu YJ, Cai ZP, Li HQ, Li MZ, He K, Gou XP, Li J. Essential roles of SERKs in the ROOT MERISTEM GROWTH FACTOR-mediated signaling pathway[J]. Plant Physiology, 2022.

https://doi.org/10.1093/plphys/kiac036

ROOT MERISTEM GROWTH FACTORs (RGFs), a group of peptide hormones, play key roles in root apical meristem development. In Arabidopsis (Arabidopsis thaliana), there are 11 members of RGFs, in which at least RGF1, RGF2, and RGF3 are expressed at the root tip and are involved in root stem cell niche maintenance. RGFs are perceived by five functionally redundant receptor-like protein kinases, RGF1 INSENSITIVE 1 (RGI1) to RGI5, to maintain the expression of two downstream APETALA 2 (AP2) transcription factor genes, PLETHORA 1 (PLT1) and PLT2, and to stabilize PLT2. RGI1 to RGI3 were also named RGF RECEPTOR 1 (RGFR1) to RGFR3, respectively. Although previous studies have suggested that BRI1ASSOCIATED RECEPTOR KINASE 1 (BAK1) and its paralogs, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs), may act as coreceptors of RGIs, comprehensive genetic and biochemical analyses have not been well documented. Here, we report that single, double, and triple mutants of SERKs show various degrees of short root phenotypes and insensitivity to exogenously applied RGF1. The interaction between RGIs and BAK1 and their mutual phosphorylation are RGF1 dependent. We also found that RGF1-induced MAPK activation relies on both RGIs and SERKs. We demonstrate that RGIs play redundant roles in regulating root apical meristem development. Therefore, we genetically and biochemically substantiated that SERKs, as coreceptors, play essential roles in the RGF1-mediated signaling pathway.

5. Liu C, Tai YL, Luo JJ, Wu YH, Zhao XK, Dong RS, Ding XP, Zhao SC, Luo LJ, Liu PD, Liu GD. Integrated multi-omics analysis provides insights into genome evolution and phosphorus deficiency adaptation in pigeonpea (Cajanus cajan)[J]. Hortic. Res., 2022, 9:uhac107.

https://doi.org/10.1093/hr/uhac107

Pigeonpea (Cajanus cajan) is an important legume food crop and plays a crucial role in a secure food supply in many developing countries. Several previous studies have suggested that pigeonpea has great potential for phosphorus (P) deficiency tolerance, but little is known about the underlying mechanism. In this study, the physiological and molecular responses of pigeonpea roots to phosphate (Pi) starvation were investigated through integrating phenotypic, genomic, transcriptomic, metabolomic, and lipidomic analyses. The results showed that low-Pi treatment increased total root length, root surface area, and root acid phosphatase activity, and promoted the secretion of organic acids (e.g. citric acids, piscidic acids, and protocatechuic acids) and the degradation of phospholipids and other P-containing metabolites in the roots of pigeonpea. Consistent with the morphological, physiological, and biochemical changes, a large number of genes involved in these Pi-starvation responses were significantly upregulated in Pi-deficient pigeonpea roots. Among these Pi-starvation response genes upregulated by low-Pi treatment, four gene families were expanded through recent tandem duplication in the pigeonpea genome, namely phosphate transporter 1 (PHT1), phosphoethanolamine/ phosphocholine phosphatase (PECP), fasciclin-like arabinogalactan protein (FLA), and glutamate decarboxylase (GAD). These gene families may be associated with Pi uptake from the soil, phospholipid recycling, root morphological remodeling, and regulation of organic acid exudation. Taken together, our results suggest that pigeonpea employs complex Pi-starvation responses to strengthen P acquisition and utilization during low-Pi stress. This study provides new insights into the genome evolution and P deficiency adaptation mechanism of pigeonpea.

3. Cai ZP, Xie ZY, Huang LY, Wang ZX, Pan M, Yu XD, Xu ST, Luo JJ. Full-length transcriptome analysis of Adiantum flabellulatum gametophyte[J]. Peer J, 2022,10: e13079.

http://doi.org/10.7717/peerj.13079

Ferns are important components of plant communities on earth, but their genomes are generally very large, with many redundant genes, making whole genome sequencing of ferns prohibitively expensive and time-consuming. This means there is a significant lack of fern reference genomes, making molecular biology research difficult. The gametophytes of ferns can survive independently, are responsible for sexual reproduction and the feeding of young sporophytes, and play an important role in the alternation of generations. For this study, we selected Adiantum flabellulatum as it has both ornamental and medicinal value and is also an indicator plant of acidic soil. The full-length transcriptome sequencing of its gametophytes was carried out using PacBio three-generation sequencing technology. A total of 354,228 transcripts were obtained, and 231,705 coding sequences (CDSs) were predicted, including 5,749 transcription factors (TFs), 2,214 transcription regulators (TRs) and 4,950 protein kinases (PKs). The transcripts annotated by non-redundant protein sequence database (NR), Kyoto encyclopedia of genes and genomes (KEGG), eukaryotic ortholog groups (KOG),Swissprot, protein family (Pfma), nucleotide sequence database (NT) and gene ontology (GO) were 251,501, 197,474, 193,630, 194,639, 195,956, 113,069 and 197,883, respectively. In addition, 138,995 simple sequence repeats (SSRs) and 111,793 long non-coding RNAs (lncRNAs) were obtained. We selected nine chlorophyll synthase genes for qRT-PCR, and the results showed that the full-length transcript sequences and the annotation information were reliable. This study can provide a reference gene set for subsequent gene expression quantification.

A proposed model of adaptive strategies to Pi deficiency in leaves and roots of elephant grass. Red indicate genes whose expression levels were up-regulated by Pi starvation. Purple and blue indicate metabolites whose accumulation increased (purple) or decreased (blue) in response to Pi deficiency.

DEGs downregulated in the BR biosynthesis pathway were in the majority in the BL treatment. (A) The BR biosynthesis pathway (https://www.kegg.jp/entry/map00905 (accessed on 24 April 2022)). (B) Expression heat maps of the DEGs in CYP90B1, CYP724B1, CYP90C1, CYP90D1, CPD, CYP92A6, BR6ox1, and BAS1 in the six samples, respectively.

Venn diagram of transcript number distribution annotated by 5 databases and homolo-gous species information in the NR database. (A) Venn diagram of transcripts annotated by NR, KEGG, KOG, Swissprot and Pfam databases. The numbers in the overlapping areas represent the numbers of shared genes. (B) The proportion of transcripts derived from homologous species and annotated by the NR database.

Amodel showing SERKs are essential coreceptors in the RGF1-RGI1-mediated signaling pathway to control root development. A, Without SERKs, RGF1-RGI1-mediated signaling pathway cannot be initiated. As a result, the transcriptions of PLT1 and PLT2 remain at a low level and the cell division activity in root tip is reduced. B, In the presence of SERKs, RGI1-BAK1 complex can be formed upon the in-duction of RGF1, the downstream signaling cascade can be activated. The expression levels of PLT1 and PLT2 are up-regulated and the pro-tein stability of PLT2 is enhanced which promotes the cell division in root tips.

Overview of genes related to phospholipid degradation. a Changes in the expression of genes encoding enzymes involved in the phospholipid degradation pathway in the roots of pigeonpea seedlings grown under +Pi versus −Pi conditions. Log2(−Pi/+Pi) of upregulated genes is shown as a heat map. PLC, phospholipase C; NPC, non-specific phospholipase C; PLD, phospholipase D; PAH, phosphatidate phosphohydrolase; PECP, phosphoethanolamine/phosphocholine phosphatase; LysoPL, lysophospholipase; GDPD, glycerophosphodiester phosphodiesterase; GPP, glycerol-3 phosphatase. b Phylogenetic tree analysis of PECPs in pigeonpea (C. cajan), soybean (G. max), barrel medic (M. truncatula), common bean (P. vulgaris), and Arabidopsis (A. thaliana). Different colored circles represent different species. c Microcollinearity of tandemly duplicated PECPs in pigeonpea compared with soybean and barrel medic. PECPs are marked with their corresponding gene IDs. d PECP activity in the roots of pigeonpea. Data are means ± standard error of three biological replicates. Asterisks indicate significant differences between −Pi and +Pi treatments according to Student’s t-test: ^∗∗.001 ≤ P < .01.

1. Cai, ZP, Huang Z, Wang ZX, Tao Y, Wu FH, Yu XD, Luo JJ. Identification of the related genes on the asymmetric root growth of Oryza sativa induced by ethylene through transcriptome sequencing, GO and KEGG analysis[J]. Acta Physiologiae Plantarum, 2021, 43(7): 99.doi:10.1007/s11738-021-03271-9.

https://doi.org/10.1007/s11738-021-03271-9

Ethylene (ETH) can induce asymmetric root growth (ARG) in plants to form waves and coiling, which can be eliminated by the ETH perception antagonist silver thiosulfate (STS), indicating that this process depends on the ETH signal transduction. Nevertheless, the related genes have not been reported. In this study, different concentrations of ethephon (ETP) or 1–aminocyclopropane–1–carboxylic acid (ACC) were applied to explore the optimal concentration for inducing the ARG of Oryza sativa. On this basis, five treatments (Water, ETP, ETP + STS, ACC, ACC + STS) were set up. Then, transcriptome sequencing, GO and KEGG analysis were used to identify the genes related to ETH-induced ARG. Finally, 1533 DEGs were screened. Among them, 43 were involved in phytohormone signaling transduction and 26 were phytohormone synthase genes. Meanwhile, 13 DEGs could regulate the polar auxin transport (PAT) and affect the tropism of roots, including the auxin influx carrier gene OsAUX2; five ATP-binding cassette (ABC) transporter family genes OsABCB4, OsABCB8, OsABCB15, OsABCB17, OsABCB21; 2 histidine kinase genes OsHK3, OsHK5; 4 flavonoid biosynthesis genes OsCHI/OsTT5, OsFLS, OsHCT1, OsCYP75B3; and the E3 ubiquitin–protein ligase gene OsWAV3. These 13 DEGs were all up-regulated by ETH. Besides, the receptor-like kinase (RLK) gene Oryza sativa Root Meander Curling (OsRMC), a negative regulator of jasmonic acid-induced ARG, was also up-regulated in ETH. The results obtained here provide a first step toward understanding the molecular mechanisms of ETH-induced ARG.

2. Cai ZP, Huang Z, Huang CJ, Jin X, Yang W, Jiang GB, Wang ZX, Wu FH, Yu XD, Luo JJ. Transcriptome analysis on asymmetric root growth of Oryza sativa Induced by brassinosteroids via ethylene pathway[J]. Journal of Plant Biology. 2021.

https://doi.org/10.1007/s12374-021-09308-3

The growth of plant roots is regulated by various factors. Brassinosteroids (BRs) can induce asymmetric root growth (ARG) to form waves and coils, and this phenomenon can be eliminated by ethylene (ETH) inhibitors aminoacetic acid (AOA) or silver thiosulfate (STS), indicating this process depends on ETH pathway. Nevertheless, the research on related genes has not been reported. In this study, four treatments (Water, Brassinolide (BL), BL + AOA, and BL + STS) were set up using the seedlings of Oryza sativa as materials. The differentially expressed genes (DEGs) in BR-induced ARG via ETH pathway were identified by transcriptome sequencing, weighted gene co-expression network analysis (WGCNA), and real-time quantitative PCR (qRT-PCR). Finally, we screened 70 DEGs, including ALPHA-EXPANSINs (OsEXPAs), WALL-ASSOCIATED RECEPTOR KINASE-LIKE 16 (OsWAKL16), and TRANSMEMBRANE KINASE (OsTMK) that regulated cell elongation and expansion, as well as plant hormone synthase genes, 1-AMINO-CYCLOPROPANE-1-CARBOXYLIC ACID OXIDASEs (OsACOs), OsYUCCAs, and CYTOKININ OXIDASE(OsCKX). Most of them were up-regulated by BRs via ETH pathway. In addition, the receptor-like kinase (RLK) gene ROOT MEADNDER CURLING (OsRMC), a negative regulator in jasmonic acid-induced ARG, could be inhibited by BRs, and this process also depended on ETH pathway. The identification of the above genes provides evidence for revealing the molecular mechanism in the process.

3. Luo JJ, Liu YX, Zhang HK, Wang JP, Chen ZJ, Luo LJ, Liu GD, Liu PD. Metabolic alterations provide insights into Stylosanthes roots responding to phosphorus deficiency[J]. BMC Plant Biol., 2020, 20:85.

https://doi.org/10.1186/s12870-020-2283-z

Phosphorus (P) deficiency is one of the major constraints limiting plant growth, especially in acid soils. Stylosanthes (stylo) is a pioneer tropical legume with excellent adaptability to low P stress, but its underlying mechanisms remain largely unknown. In this study, the physiological, molecular and metabolic changes in stylo responding to phosphate (Pi) starvation were investigated. Under low P condition, the growth of stylo root was enhanced, which was attributed to the up-regulation of expansin genes participating in root growth. Metabolic profiling analysis showed that a total of 256 metabolites with differential accumulations were identified in stylo roots response to P deficiency, which mainly included flavonoids, sugars, nucleotides, amino acids, phenylpropanoids and phenylamides. P deficiency led to significant reduction in the accumulation of phosphorylated metabolites (e.g., P-containing sugars, nucleotides and cholines), suggesting that internal P utilization was enhanced in stylo roots subjected to low P stress. However, flavonoid metabolites, such as kaempferol, daidzein and their glycoside derivatives, were increased in P-deficient stylo roots. Furthermore, the qRT-PCR analysis showed that a set of genes involved in flavonoids synthesis were found to be up-regulated by Pi starvation in stylo roots. In addition, the abundances of phenolic acids and phenylamides were significantly increased in stylo roots during P deficiency. The increased accumulation of the metabolites in stylo roots, such as flavonoids, phenolic acids and phenylamides, might facilitate P solubilization and cooperate with beneficial microorganisms in rhizosphere, and thus contributing to P acquisition and utilization in stylo.   These results suggest that stylo plants cope with P deficiency by modulating root morphology, scavenging internal Pi from phosphorylated metabolites and increasing accumulation of flavonoids, phenolic acids and phenylamides. This study provides valuable insights into the complex responses and adaptive mechanisms of stylo roots to P deficiency.

A model describe the ARG regulated by ETH signaling pathway

A model describes ARG regulated by BRs via ETH pathway. According to the results of transcriptome sequencing, there were at least two processes in BRs-induced ARG via ETH pathway.

Flavonoid metabolism pathway in stylo roots response to P deficiency. The pathway was designed based on KEGG pathways (http://www.kegg.jp/kegg/pathway.html)as described by Zhang et al. [51]and Kc et al. [43]. The light red, blue, yellow and grey colors indicate up-regulated,down-regulated, no response and no identified metabolites in metabolome, respectively. The solid arrows show direct and dotted arrows represent indirect and speculated steps in the pathway. Important genes are marked on the metabolic procedure.

1. Li HQ, Cai ZP, Wang XJ, Li MZ, Cui YW, Cui N, Yang F, Zhu MS, Zhao JX, Du WB, He K, Yi J, Tax F. E., Hou SW, Li J, Gou XP. SERK receptor-like kinases control division patterns of vascular precursors and ground tissue stem cells during embryo development in Arabidopsis[J]. Molecular Plant, 2019, 12(7): 984-1002.

https://doi.org/10.1016/j.molp.2019.04.011

During embryo development, the vascular precursors and ground tissue stem cells divide to renew themselves and produce the vascular tissue, endodermal cells, and cortical cells. However, the molecular mechanisms regulating division of these stem cells have remained largely elusive. In this study, we show that loss of function of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes results in aberrant embryo development. Fewer cortical, endodermal, and vascular cells are generated in the embryos of serk1 serk2 bak1 triple mutants. WUSCHEL-RELATED HOMEOBOX 5 (WOX5) is ectopically expressed in vascular cells of serk1 serk2 bak1 embryos. The first transverse division of vascular precursors in mid-globular embryos and second asymmetric division of ground tissue stem cells in early-heart embryos are abnormally altered to a longitudinal division. The embryo defects can be partially rescued by constitutively activated mitogen-activated protein kinase (MAPK) kinase kinase YODA (YDA) and MAPK kinase MKK5. Taken together, our results reveal that SERK-mediated signals regulate division patterns of vascular precursors and ground tissue stem cells, likely via the YDA-MKK4/5 cascade, during embryo development.

2. Cai ZP, Yang W, Zhang HY, Luo JJ, Wu FH, Jing X, Meng SL, Wang XY, Yu XD. Ethylene participates in the brassinolide-regulated asymmetric growth of O. sativa root[J]. South African Journal of Botany, 2018, 119: 86-93.

https://doi.org/10.1016/j.sajb.2018.08.017

In this work, brassinosteroid (BR)-induced root asymmetric growth as well as its interaction with ethylene (ETH) were investigated. The results reveal that 2, 4-epibrassinolide (2,4-eBL) can induce asymmetric growth of the primary roots of monocotyledonous Oryza sativa to form root coils and waves under both illumination and dark conditions. Similarly, ETH can also induce asymmetric growth of the primary roots of O. sativa to form hooked structures. Upon adding a concentration of 2 × 10⁻⁸ M 2,4-eBL and inhibitors of ethylene simultaneously, silver thiosulfate (STS) and aminooxyacetic acid could completely eliminate the 2,4-eBL-induced asymmetric growth of roots, while the asymmetric growth could also be partly eliminated by CoCl2. Meanwhile, the effect of ETH inhibitors was proven to be reversible, wherein 2,4-eBL can exert its function and induce the asymmetric growth of O. sativa roots after the removal of STS. These conclusions provide a scientific foundation for further investigations of the relationships between BR and ETH.

3. Jiang SJ, Liu YJ, Luo JJ, Qin MS, Johnson NC, Öpik M, Vasar M, Chai YX, Zhou XL, Mao L, Du GZ, An LZ, Feng HY. Dynamics of arbuscular mycorrhizal fungal community structure and functioning along a nitrogen enrichment gradient in an alpine meadow ecosystem[J]. New Phytol., 2018, 220(4):1222-1235.

https://doi.org/10.1111/nph.15112

Nitrogen (N) availability is increasing dramatically in many ecosystems, but the influence of elevated N on the functioning of arbuscular mycorrhizal (AM) fungi in natural ecosystems is not well understood. We measured AM fungal community structure and mycorrhizal function simultaneously across an experimental N addition gradient in an alpine meadow that is limited by N but not by phosphorus (P). AM fungal communities at both whole-plant-community (mixed roots) and single-plant-species (Elymus nutans roots) scales were described using pyro-sequencing, and the mycorrhizal functioning was quantified using a mycorrhizal-suppression treatment in the field (whole-plant-community scale) and a glasshouse inoculation experiment (singleplant-species scale). Nitrogen enrichment progressively reduced AM fungal abundance, changed AM fungal community composition, and shifted mycorrhizal functioning towards parasitism at both whole-plant-community and E.nutans scales. N-induced shifts in AM fungal community composition were tightly linked to soil N availability and/or plant species richness, whereas the shifts in mycorrhizal function were associated with the communities of specific AM fungal lineages. The observed changes in both AM fungal community structure and functioning across an N enrichment gradient highlight that N enrichment of ecosystems that are not P-limited can induce parasitic mycorrhizal functioning and influence plant community structure and ecosystem sustainability.

4. Cai ZP, Jing X, Tian XH, Jing JL, Liu F, Wang XY. Direct and indirect in vitro, plant regeneration and the effect of brassinolide on callus, differentiation of Populus euphratica, Oliv[J]. South African Journal of Botany, 2015, 97: 143-148.

https://doi.org/10.1016/j.sajb.2015.01.006

Populus euphratica Oliver is a poplar species with very strong tolerance to drought, high temperature, alkalinity and salinity, but it is difficult to propagate due to its intrinsic physiological defects. In this study, an efficient in vitro plant regeneration protocol for P. euphratica has been established by using the young leaves and stems as explants. With this protocol, among the six types of explants tested, five ones simultaneously produced shoots and calli on the MS medium supplemented with 0.5 mg/L c-naphthalene acetic acid (NAA) and 0.5 mg/L N^6_benzyl aminopurine (BA). Besides, the calli produced further differentiated into adventitious buds or shoots on the MS medium supplemented with 0.1 mg/L NAA, 0.5 mg/L BA and 10^-4-10^-1 mg/L brassinolide (BL). The regeneration rates of buds/shoots that differentiated from the explants and calli were up to 52.7% and 73.3% respectively. Up to 75% of the shoots rooted on the 1/2 MS medium with indole-3-butyric acid (IBA) and grew well in outdoor soil. Moreover, it was found that in the presence of BL, the shoot regeneration rate of callus was increased by 4-57%, and the time for shoot regeneration was shortened by 20-30 days. This is the first report that describes direct and indirect organogenesis simultaneously occurring in P. euphratica, and that BL can significantly promote the differentiation of P. euphratica calli into shoots.

5. Wu HJ, Liu XH, Chen K, Cai ZP, Luo XJ, Zhang T, Wang XY. Disintegration of microsporocytes in a male sterile mutant of Brassica napus L. is possibly associated with endoplasmic reticulum-dependent autophagic programmed cell death[J]. Euphytica, 2009, 170(3): 263-274. ‍

https://link.springer.com/article/10.1007/s10681-009-9977-5

The mechanism of anther abortion in a male sterile (ms) line (Longyou 9S) of Brassica napus L. was evaluated by a combination of light and electron microscopies. Light microscopy showed that the tapetal cells of the ms line appeared smaller than those of its fertile line, sporogenous cells were rich in vacuoles, and pollen mother cells dismantled before the tetrad stage. Electron microscopy demonstrated that sporogenous cells were also rich in very long, plate-shaped endoplasmic reticulum cisternae that actively encircled portions of cytoplasm with organelles. In later stages, many large membrane-bound inclusions were observed in the sporogenous cells. These membrane-bound inclusions were lined by single or multiple layer(s) of the endoplasmic reticulum membranes and contained vacuoles, ribosome, plastid, mitochondria, small membrane-bound inclusion, and/or intact nuclei. In meiotic interphase and early meiosis prophase I, many vesicle aggregations and multivesicular bodies containing numerous vesicles appeared in pollen mother cells. More interestingly, some of the multivesicular bodies lay in deep cytoplasm or near cell wall, and some appeared fused with plasmalemma and released the inner vesicles out of plasmalemma. Such released vesicles gradually dispersed and later disappeared. The observations suggest that active endoplasmic reticulum-dependent autophagic programmed cell death and multivesicular body-dependent polar vesicle trafficking are probably present in the studied male sterile line.

(A–E) Defective embryo development of mkk4-18 mkk5-18/+ mutants. Compared with wild-type-like embryos (A),3.2% of progeny from mkk4-18 mkk5-18/+ mutants bend in reverse (B).15.5% of embryos display weak defects with shorter cotyledons and hypocotyls (C).1.6% of embryos show severe defects (D),and 2% of embryos are outside of the developing young seeds (E). (F and G) WOX5 expression is elevated in the vascular tissue of mkk4-18 mkk5-18 embryos (G) compared with wild-type-like embryos (F). (H–J) Constitutively activated MKK5 (MKK5^DD)partially suppresses the defective embryo development of serk1-8 serk2-1 bak1-4 mutants. A homo-zygous MKK5^DD transgene was driven by SERK1 promoter in serk1-8/+serk2-1 bak1-4 plants. 77.1% of embryos show wild-type-like phenotypes (H). 18.3% of embryos show a partially suppressed phenotype, which eventually can fill the seeds (I).The remaining embryos show defects the same as in the serk1-8 serk2-1 bak1-4 mutants (J). (K–R) Division defects of the ground tissue stem cells (red stars) are partially restored in serk1-8 serk2-1 bak1-4 embryos by MKK5^DD.75.9% of embryos segregated from serk1-8/+serk2-1 bak1-4 mutants harboring a homozygous transgene of MKK5^DD show wild-type-like phenotypes (K and L). The ground tissue stem cells exist to produce daughter cells in 2.3% of embryos (M and N).Part of the ground tissue stem cells exist in 9.3% of embryos (O and P).No ground tissue stem cells can be found in 12.5% of embryos (Q and R).Divisions of the ground tissue stem cells are indicated by white (normal) or red (abnormal) arrows. The orange rectangle-enclosed part of the embryo in (K), (M), (O),and (Q) is enlarged in (L), (N), (P), and (R), respectively.(S) Amodel for SERKs in regulating embryo development. SERKs may function together with an unknown RLK to maintain the identity of the vascular precursors and the ground tissue stem cells partially via the YDA-MKK4/5 cascade. "n" indicates the total number of examined embryos. Cell outlines are marked with FM4-64 in (F), (G),and (K) to (R).Scale bars, 100 mm (A–E and H–J) and 10 mm (F, G,and K–R).

2,4-eBL induced the asymmetric growth of O. sativa primary roots. (A and B) Natural growth of O. sativa primary roots; (C and D) wave formation in O. sativa primary roots with 2×10^-8 M 2,4-eBL; (E and F) coiling formation in O. sativa primary roots with 2 × 10⁻⁸ M2,4-eBL; (G) wave formation before coiling formation in O.sativa primary roots with 2 × 10⁻⁸ M2,4-eBL; (H) coiling formation before wave formation in O.   sativa primary roots with of 2 × 10⁻⁸ M2,4-eBL; (I, J, and K) percentage of 2,4-eBL-induced asymmetric growth of O. sativa primary roots. The culture time was 7 d. (A, C, and E) Culture in illumination; (B, D, and F) culture in dark. Bars = 5 mm.

Nonmetric multidimensional scaling analysis (NMDS) (Bray-Curtis distance) of community composition of arbuscular mycorrhizal (AM) fungal virtual taxa colonizing (a) mixed plant roots and (b) Elymus nutans roots among N treatments in the field experiment. N0, N5, N10 and N15 represent fertilizer applications of 0, 5, 10 and 15 g N m^-2 yr^-1,respectively. Ellipses with different colors indicate 95% confidence intervals for each treatment. Significant plant and/or soil variables that are correlated with each community ordination are shown.

Root formation of the regenerated shoots. A and B are the green shoots that regenerated from explants rooted on rooting medium; C, D and E are the green shoots that regenerated from calli rooted on rooting medium; F, G and H are the magnification of the regenerated roots, showing dense root hairs. I, J and K are the white shoots that regenerated from explants that became green in the light, but did not root. L–O are the plantlets transplanted to soil. Bars = 2 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Transversal anther sections of the ms line. a–c SC stage: anther wall has an even epidermis and very poorly developed tapetum; the SCs are rich in vacuole-like or ganelles andhave a nucleus with large nucleolus. d Enlarged view of the SCs in c,showing abundant vacuole-like structures. e–i Early meiosis prophase: large patches of gaps occur between the PMCs, and much TB-stained substance has accumulated. j–l Both the tapetum and PMCs have collapsed, the central sacs contain much amorphous structures and TB-stained matrix, but the epidermis, endothecium, and middle layer of anther wall appear intact. Bar 5 μm for all images.