Genome-wide analysis of the KNOX gene family in Moso bamboo: insights into their role in promoting the rapid shoot growth.

BACKGROUND: KNOTTED1-like homeobox (KNOX) genes, plant-specific homologous box transcription factors (TFs), play a central role in regulating plant growth, development, organ formation, and response to biotic and abiotic stresses. However, a comprehensive genome-wide identification of the KNOX genes in Moso bamboo (Phyllostachys edulis), the fastest growing plant, has not yet been conducted, and the specific biological functions of this family remain unknown. RESULTS: The expression profiles of 24 KNOX genes, divided into two subfamilies, were determined by integrating Moso bamboo genome and its transcriptional data. The KNOX gene promoters were found to contain several light and stress-related cis-acting elements. Synteny analysis revealed stronger similarity with rice KNOX genes than with Arabidopsis KNOX genes. Additionally, several conserved structural domains and motifs were identified in the KNOX proteins. The expansion of the KNOX gene family was primarily regulated by tandem duplications. Furthermore, the KNOX genes were responsive to naphthaleneacetic acid (NAA) and gibberellin (GA) hormones, exhibiting distinct temporal expression patterns in four different organs of Moso bamboo. Short Time-series Expression Miner (STEM) analysis and quantitative real-time PCR (qRT-PCR) assays demonstrated that PeKNOX genes may play a role in promoting rapid shoot growth. Additionally, Gene Ontology (GO) and Protein-Protein Interaction (PPI) network enrichment analyses revealed several functional annotations for PeKNOXs. By regulating downstream target genes, PeKNOXs are involved in the synthesis of AUX /IAA, ultimately affecting cell division and elongation. CONCLUSIONS: In the present study, we identified and characterized a total of 24 KNOX genes in Moso bamboo and investigated their physiological properties and conserved structural domains. To understand their functional roles, we conducted an analysis of gene expression profiles using STEM and RNA-seq data. This analysis successfully revealed regulatory networks of the KNOX genes, involving both upstream and downstream genes. Furthermore, the KNOX genes are involved in the AUX/IAA metabolic pathway, which accelerates shoot growth by influencing downstream target genes. These results provide a theoretical foundation for studying the molecular mechanisms underlying the rapid growth and establish the groundwork for future research into the functions and transcriptional regulatory networks of the KNOX gene family.

Seed Germination and Growth Improvement for Early Maturing Pear Breeding.

Breeding early maturing cultivars is one of the most important objectives in pear breeding. Very early maturing pears provide an excellent parental material for crossing, but the immature embryo and low seed germination of their hybrid progenies often limit the selection and breeding of new early maturing pear cultivars. In this study, we choose a very early maturing pear cultivar 'Pearl Pear' as the study object and investigate the effects of cold stratification, the culture medium, and the seed coat on the germination and growth of early maturing pear seeds. Our results show that cold stratification (4 °C) treatment could significantly improve the germination rates of early maturing pear seeds. A total of 100 days of cold-temperature treatment in 4 °C and in vitro germination on White medium increased the germination rate to 84.54%. We also observed that seed coat removal improved the germination of early maturing pear seeds, with middle seed coat removal representing the optimal method, with a high germination rate and low contamination. The results of our study led to the establishment of an improved protocol for the germination of early maturing pear, which will greatly facilitate the breeding of new very early maturing pear cultivars.

Genome-Wide Identification of SPX Family Genes and Functional Characterization of PeSPX6 and PeSPX-MFS2 in Response to Low Phosphorus in Phyllostachys edulis.

Moso bamboo (Phyllostachys edulis) is the most widely distributed bamboo species in the subtropical regions of China. Due to the fast-growing characteristics of P. edulis, its growth requires high nutrients, including phosphorus. Previous studies have shown that SPX proteins play key roles in phosphorus signaling and homeostasis. However, the systematic identification, molecular characterization, and functional characterization of the SPX gene family have rarely been reported in P. edulis. In this study, 23 SPXs were identified and phylogenetic analysis showed that they were classified into three groups and distributed on 13 chromosomes. The analysis of conserved domains indicated that there was a high similarity between PeSPXs among SPX proteins in other species. RNA sequencing and qRT-PCR analysis indicated that PeSPX6 and PeSPX-MFS2, which were highly expressed in roots, were clearly upregulated under low phosphorus. Co-expression network analysis and a dual luciferase experiment in tobacco showed that PeWRKY6 positively regulated the PeSPX6 expression, while PeCIGR1-2, PeMYB20, PeWRKY6, and PeWRKY53 positively regulated the PeSPX-MFS2 expression. Overall, these results provide a basis for the identification of SPX genes in P. edulis and further exploration of their functions in mediating low phosphorus responses.

Genome-Wide Identification of the Highly Conserved INDETERMINATE DOMAIN (IDD) Zinc Finger Gene Family in Moso Bamboo (Phyllostachys edulis).

INDETERMINATE DOMAIN (IDD) proteins, a family of transcription factors unique to plants, function in multiple developmental processes. Although the IDD gene family has been identified in many plants, little is known about it in moso bamboo. In this present study, we identified 32 PheIDD family genes in moso bamboo and randomly sequenced the full-length open reading frames (ORFs) of ten PheIDDs. All PheIDDs shared a highly conserved IDD domain that contained two canonical C2H2-ZFs, two C2HC-ZFs, and a nuclear localization signal. Collinearity analysis showed that segmental duplication events played an important role in expansion of the PheIDD gene family. Synteny analysis indicated that 30 PheIDD genes were orthologous to those of rice (Oryza sativa). Thirty PheIDDs were expressed at low levels, and most PheIDDs exhibited characteristic organ-specific expression patterns. Despite their diverse expression patterns in response to exogenous plant hormones, 8 and 22 PheIDDs responded rapidly to IAA and 6-BA treatments, respectively. The expression levels of 23 PheIDDs were closely related to the outgrowth of aboveground branches and 20 PheIDDs were closely related to the awakening of underground dormant buds. In addition, we found that the PheIDD21 gene generated two products by alternative splicing. Both isoforms interacted with PheDELLA and PheSCL3. Furthermore, both isoforms could bind to the cis-elements of three genes (PH02Gene17121, PH02Gene35441, PH02Gene11386). Taken together, our work provides valuable information for studying the molecular breeding mechanism of lateral organ development in moso bamboo.

Diverse responses of pqqC- and phoD-harbouring bacterial communities to variation in soil properties of Moso bamboo forests.

Phosphate-mobilizing bacteria (PMB) play a critical role in the regulation of phosphorus availability in the soil. The microbial genes pqqC and phoD encode pyrroloquinoline quinone synthase and bacterial alkaline phosphatase, respectively, which regulate inorganic and organic phosphorus mobilization, and are therefore used as PMB markers. We examined the effects of soil properties in three Moso bamboo forest sites on the PMB communities that were profiled using high-throughput sequencing. We observed differentiated responses of pqqC- and phoD-harbouring PMB communities to various soil conditions. There was significant variation among the sites in the diversity and structure of the phoD-harbouring community, which correlated with variation in phosphorus levels and non-capillary porosity; soil organic carbon and soil water content also affected the structure of the phoD-harbouring community. However, no significant difference in the diversity of pqqC-harbouring community was observed among different sites, while the structure of the pqqC-harbouring bacteria community was affected by soil organic carbon and soil total nitrogen, but not soil phosphorus levels. Overall, changes in soil conditions affected the phoD-harbouring community more than the pqqC-harbouring community. These findings provide a new insight to explore the effects of soil conditions on microbial communities that solubilize inorganic phosphate and mineralize organic phosphate.

Screening and activity assessing of phosphorus availability improving microorganisms associated with bamboo rhizosphere in subtropical China.

Phosphorus-solubilizing microorganisms (PSMicros) play vital roles in helping plants to resist phosphorus (P) deficiency in soils, while their activities may vary with site conditions. The present study investigated the microbial diversity and subsequently screened PSMicro strains from rhizosphere soils at five bamboo forests in subtropical China, among which four were developed in a same stand. The activities of the screened PSMicros were also assessed. The results showed great variation in microbial diversity among different forests. Concomitantly, a total of 52 PSMicro strains were isolated and identified to 10 bacterial genera and 4 fungal genera, with different forest rhizosphere soils containing different PSMicros and/or showing different abundances for a certain PSMicro genus, despite some PSMicros would not grow readily on plates. Different, and even the same microbial genera isolated across the five forests, varied significantly in the amount of P that they solubilized from the medium, which ranged from 18.5 to 581.33 mg L-1 . Among the isolated PSMicros, species of Bacillus, Kluyvera, Buttiauxella, Meyerozyma and Penicillium were preponderant to liberate P from organic and inorganic P pools. This will have implications for biotechnological exploitation of microbes to alleviate P limitation in agricultural and natural systems with a sustainable green ecological approach.

Comparative Analysis of the Lignification Process of Two Bamboo Shoots Stored at Room Temperature.

Two types of bamboo shoots, high bamboo (Phyllostachys prominens) shoots (HBSes) and moso bamboo (Phyllostachys edulis) shoots (MBSes), underwent a fast post-harvest lignification process under room temperature storage. To explore the mechanism of lignification in two types of bamboo shoots after post-harvest during room temperature storage, the measurement of cell wall polymers (lignin and cellulose) and enzyme activities of phenylalanine ammonialyase (PAL) and peroxidase (POD), and relative expression of related transcription networks factors (TFs) were performed. The results suggested that the lignification process in HBSes is faster than that in MBSes because of incremental increase in lignin and cellulose contents within 6 days and the shorter shelf-life. Additionally, compared with the expression pattern of lignification-related TFs and correlation analysis of lignin and cellulose contents, MYB20, MYB43, MYB85 could function positively in the lignification process of two types of bamboo shoots. A negative regulator, KNAT7, could negatively regulate the lignin biosynthesis in two types of bamboo shoots. In addition, MYB63 could function positively in HBSes, and NST1 could function negatively in MBSes. Notably, MYB42 may function differently in the two types of bamboo shoots, that is, a positive regulator in HBSes, but a negative regulator in MBSes. Transcription networks provide a comprehensive analysis to explore the mechanism of lignification in two types of bamboo shoots after post-harvest during room temperature storage. These results suggest that the lignification of bamboo shoots was mainly due to the increased activity of POD, higher expression levels of MYB20, MYB43, MYB63, and MYB85 genes, and lower expression levels of KNAT7 and NST1 genes, and the lignification process of HBSes and MBSes had significant differences.

The acid invertase gene family is involved in internode elongation in Phyllostachys heterocycla cv. pubescens.

Acid invertases (INVs) play a pivotal role in both vegetative and reproductive growth of plants. However, their possible functions in fast-growing plants such as bamboo are largely unknown. Here, we report the molecular characterization of acid INVs in Phyllostachys heterocycla cv. pubescens, a fast-growing bamboo species commercially grown worldwide. Nine acid INVs (PhINVs), including seven cell wall INVs (PhCWINV1, PhCWINV2, PhCWINV3, PhCWINV4, PhCWINV5, PhCWINV6 and PhCWINV7) and two vacuolar INVs (PhVINV11 and PhVINV12) were isolated. Bioinformatic analyses demonstrated that they all share high amino acid identity with other INVs from different plant species and contain the motifs typically conserved in acid INV. Enzyme activity assays revealed a significantly higher INV activity in the fast-growing tissues, such as the elongating internodes of stems. Detailed quantitative reverse-transcription PCR analyses showed various expression patterns of PhINVs at different developmental stages of the elongating stems. With the exception of PhCWINV6, all PhINVs were ubiquitously expressed in a developmental-specific manner. Further studies in Arabidopsis exhibited that constitutive expression of PhCWINV1, PhCWINV4 or PhCWINV7 increased the biomass production of transgenic plants, as indicated by augmented plant heights and shoot dry weights than the wild-type plants. All these results suggest that acid INVs play a crucial role in the internode elongation of P. heterocycla cv. pubescens and would provide valuable information for the dissection of their exact biological functions in the fast growth of bamboo.

Transcriptome analysis provides insights into xylogenesis formation in Moso bamboo (Phyllostachys edulis) shoot.

Maturation-related changes in cell wall composition and the molecular mechanisms underlying cell wall changes were investigated from the apical, middle and basal segments in moso bamboo shoot (MBS). With maturation extent from apical to basal regions in MBS, lignin and cellulose content increased, whereas heteroxylan exhibited a decreasing trend. Activities of phenylalanine amonnialyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and cinnamate-4-hydroxylase (C4H), which are involved in lignin biosynthesis, increased rapidly from the apex to the base sections. The comparative transcriptomic analysis was carried out to identify some key genes involved in secondary cell walls (SCW) formation underlying the cell wall compositions changes including 63, 8, 18, and 31 functional unigenes encoding biosynthesis of lignin, cellulose, xylan and NAC-MYB-based transcription factors, respectively. Genes related to secondary cell wall formation and lignin biosynthesis had higher expression levels in the middle and basal segments compared to those in the apical segments. Furthermore, the expression profile of PePAL gene showed positive relationships with cellulose-related gene PeCESA4, xylan-related genes PeIRX9 and PeIRX10. Our results indicated that lignification occurred in the more mature middle and basal segments in MBS at harvest while lignification of MBS were correlated with higher expression levels of PeCESA4, PeIRX9 and PeIRX10 genes.

Molecular Mechanism of Xylogenesis in Moso Bamboo (Phyllostachys edulis) Shoots during Cold Storage.

A bamboo shoot is the immature stem of the woody grass and a nutritious and popular vegetable in East Asia. However, it undergoes a rapid xylogenesis process right after harvest, even being stored in a cold chamber. To investigate the molecular regulation mechanisms of xylogenesis in Moso bamboo (Phyllostachys edulis) shoots (MBSes) during cold storage, the measurement of cell wall polymers (cellulose, hemicellulose, and lignin) and related enzyme activities (phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), peroxidase (POD), and xylan xylosyltransferase (XylT)) and transcriptomic analysis were performed during cold storage. It was noticed that cellulose and lignin contents increased, while hemicellulose content exhibited a downward trend. PAL, CAD, and POD activity presented an upward trend generally in MBS when stored at 4 °C for 16 days. XylT activity showed a descending trend during the stages of storage, but slightly increased during the 8th to 12th days after harvest at 4 °C. Transcriptomic analysis identified 72, 28, 44, and 31 functional unigenes encoding lignin, cellulose, xylan biosynthesis enzymes, and transcription factors (TFs), respectively. Many of these secondary cell wall (SCW)-related genes showed higher expression levels in the later period of cold storage. Quantitative RT-PCR analysis of the selected genes conformed to the expression pattern. Our study provides a comprehensive analysis of MBS secondary wall biosynthesis at the molecular level during the cold storage process. The results give insight into the xylogenesis process of this economically important vegetable and shed light on solving this problem of the post-harvest industry.

Nitrogen Deposition Enhances Photosynthesis in Moso Bamboo but Increases Susceptibility to Other Stress Factors.

Atmospheric nitrogen (N) deposition can increase the susceptibility of vascular plants to other stresses, but the physiological basis of such a response remains poorly understood. This study was designed to clarify the physiological mechanisms and to evaluate bioindicators of N deposition impact on vascular plants. We evaluate multiple physiological responses to ~4 years of simulated additional N deposition (30-90 kg N ha-1 year-1) on three age-classes (1a, 3a, and 5a) of Moso bamboo. A saturating response to the additional N deposition was found both in foliar N concentration and in Pn. However, 3- and 5-year-old bamboo seemed to be less tolerant to extremely high N deposition than 1-year-old bamboo since they were saturated at a lower N addition. Furthermore, C/N/P stoichiometric ratios were very sensitive to N deposition in all three-age classes of bamboo, but the responses to N deposition in the various age-classes were diverse. We also found that the highest additional N deposition suppressed stomatal conductance and transpiration rate, suggesting an induced water stress. The stress induced by the high N load was also seen in photochemistry, where it reduced potential and actual photosynthetic use of light energy, diminished photo-protection capacity, and increased risk of the photo-damage. High additional N deposition contributed to a decrease in the foliar soluble protein contents and to an increase in the peroxidase activity (POD). Our study suggested, for the first time, that although the photosynthetic rate was enhanced by the increased N deposition in Moso bamboo, long-term high N load causes negative effects, such as damage to photosystem II. In Moso bamboo photochemical parameters are more sensitive to N deposition than photosynthetic rate or foliar N concentration. Furthermore, plant age should be taken into account when assessing plants' susceptibility to changes in global change drivers, such as N deposition. These findings facilitate the revealing of the risks potentially caused to vascular plants by increased N deposition before any visible symptoms of injury are seen.

Growth, physiological, and biochemical responses of Camptotheca acuminata seedlings to different light environments.

Light intensity critically affects plant growth. Camptotheca acuminata is a light-demanding species, but its optimum light intensity is not known. To investigate the response of C. acuminata seedlings to different light intensities, specifically 100% irradiance (PAR, 1500 ± 30 µmol m(-2) s(-1)), 75% irradiance, 50% irradiance, and 25% irradiance, a pot experiment was conducted to analyze growth parameters, photosynthetic pigments, gas exchange, chlorophyll fluorescence, stomatal structure and density, chloroplast ultrastructure, ROS concentrations, and antioxidant activities. Plants grown under 75% irradiance had significantly higher total biomass, seedling height, ground diameter, photosynthetic capacity, photochemical efficiency, and photochemical quenching than those grown under 100%, 25%, and 50% irradiance. Malondialdehyde (MDA) content, relative electrolyte conductivity (REC), superoxide anion (O(.-) 2) production, and peroxide (H2O2) content were lower under 75% irradiance. The less pronounced plant growth under 100% and 25% irradiance was associated with a decline in photosynthetic capacity and photochemical efficiency, with increases in the activity of specific antioxidants (i.e., superoxidase dismutase, peroxidase, and catalase), and with increases in MDA content and REC. Lower levels of irradiance were associated with significantly higher concentrations of chlorophyll (Chl) a and b and lower Chla/b ratios. Stomatal development was most pronounced under 75% irradiance. Modification of chloroplast development was found to be an important mechanism of responding to different light intensities in C. acuminata. The results indicated that 75% irradiance is optimal for the growth of C. acuminata seedlings. The improvement in C. acuminata growth under 75% irradiance was attributable to increased photosynthesis, less accumulation of ROS, and the maintenance of the stomatal and chloroplast structure.

Effect of silicate supplementation on the alleviation of arsenite toxicity in 93-11 (Oryza sativa L. indica).

Chronic exposure to arsenic (As) in rice has raised many health and environmental problems. As reported, great variation exists among different rice genotypes in As uptake, translocation, and accumulation. Under hydroponic culture, we find that the Chinese wild rice (Oryza rufipogon; acc. 104624) takes up the most arsenic among tested genotypes. Of the cultivated rice, the indica cv. 93-11 has the lowest arsenic translocation factor value but accumulates the maximum concentration of arsenic followed by Nipponbare, Minghui 86, and Zhonghua 11. Higher level of arsenite concentration (50 µM) can induce extensive photosynthesis and root growth inhibition, and cause severe oxidative stress. Interestingly, external silicate (Si) supplementation has significantly increased the net photosynthetic rate, and promoted root elongation, as well as strongly ameliorated the oxidative stress by increasing the activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and peroxidase in roots and/or leaves of 93-11 seedlings. Notably, 1.873 mM concentration of Si considerably decreases the total As uptake and As content in roots, but significantly increases the As translocation from roots to shoots. In contrast, Si supplementation with 1.0 mM concentration significantly increases the total As uptake and As concentrations in roots and shoots of 93-11 seedlings after 50 µM arsenite treatment for 6 days.

Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

KEY MESSAGE : We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae. Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided ( http://www.sicau.edu.cn/web/yms/PCOSWeb/PCOS.html ).