Selection and Validation of Reference Genes in Dendrocalamus brandisii for Quantitative Real-Time PCR.

Dendrocalamus brandisii (Munro) Kurz is a sympodial bamboo species with a wide distribution in tropical and subtropical regions. Due to its remarkable regenerative ability and exceptional flavor, this species plays a pivotal role in bolstering the economies of numerous nations across these regions. We recently published a high-quality genome of this species. To date, no study results have identified the optimal reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) normalization in Dendrocalamus brandisii. qRT-PCR offers a highly accurate and effective approach to analyzing gene expression. However, the precision of the resulting quantitative data hinges on the correct choice of reference genes. Twenty-one potential reference genes were identified from the D. brandisii transcriptomes. Their expression in 23 samples, including 8 different tissue organs and 15 samples of D. brandisii under various treatment conditions, were evaluated through qRT-PCR. Subsequently, four software programs-Delta CT, geNorm, NormFinder, and RefFinder-were employed to compare their expression stability. The results revealed that the selection of optimal reference genes varied based on the particular organ and condition being examined. EF-1-α-2 consistently exhibits the most stable expression across diverse tissues, while ACTIN-1, TUBULIN-1, and EF-1-α-2 were the most consistent reference genes in roots, culms, and leaves under various treatments, respectively. In this study, we identified and characterized appropriate internal genes utilized for calibrating qRT-PCR analyses of D. brandisii across different tissue organs and under various treatments. This research will provide key insights for advancing the study of gene functionality and molecular biology in D. brandisii and related species.

Conservation and Divergence of PEPC Gene Family in Different Ploidy Bamboos.

Phosphoenolpyruvate carboxylase (PEPC), as a necessary enzyme for higher plants to participate in photosynthesis, plays a key role in photosynthetic carbon metabolism and the stress response. However, the molecular biology of the PEPC family of Bambusoideae has been poorly studied, and the function of its members in the growth and development of Bambusoideae is still unclear. Here, we identified a total of 62 PEPC family members in bamboo. All the PEPC genes in the bamboo subfamily were divided into twelve groups, each group typically containing significantly fewer PEPC members in Olyra latifolia than in Phyllostachys edulis, Dendrocalamus latiflorus and Dendrocalamus brandisii. The results of an intraspecific and interspecies collinearity analysis showed that fragment replication and whole genome replication were the main driving forces of bamboo PEPC members. Furthermore, the Ka/Ks values of collinear genes showed that bamboo PEPC experienced purification selection. In addition, the promoter region of PEPC genes contains cis-acting elements related to light response, plant hormone response and response to stress. An analysis of the expression levels of the PEPC family in different developmental stages and tissues of bamboo shoots has shown that PhePEPC7, PhePEPC9 and PhePEPC10 were highly expressed in the leaves of non-flowering plants and culms. Furthermore, PhePEPC6 was significantly upregulated in leaves after GA treatment. Further research has shown that PhePEPC6 was mainly localized in the cell membrane. This provides a solid bioinformatics foundation for further understanding the biological functions of the bamboo PEPC family.

Uncovering PheCLE1 and PheCLE10 Promoting Root Development Based on Genome-Wide Analysis.

Moso bamboo (Phyllostachys edulis), renowned for its rapid growth, is attributed to the dynamic changes in its apical meristem. The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) family genes are known to play crucial roles in regulating meristem and organ formation in model plants, but their functions in Moso bamboo remain unclear. Here, we conducted a genome-wide identification of the CLE gene family of Moso bamboo and investigated their gene structure, chromosomal localization, evolutionary relationships, and expression patterns. A total of 11 PheCLE genes were identified, all of which contained a conserved CLE peptide core functional motif (Motif 1) at their C-termini. Based on Arabidopsis classification criteria, these genes were predominantly distributed in Groups A-C. Collinearity analysis unveiled significant synteny among CLE genes in Moso bamboo, rice, and maize, implying potential functional conservation during monocot evolution. Transcriptomic analysis showed significant expression of these genes in the apical tissues of Moso bamboo, including root tips, shoot tips, rhizome buds, and flower buds. Particularly, single-cell transcriptomic data and in situ hybridization further corroborated the heightened expression of PheCLE1 and PheCLE10 in the apical tissue of basal roots. Additionally, the overexpression of PheCLE1 and PheCLE10 in rice markedly promoted root growth. PheCLE1 and PheCLE10 were both located on the cell membrane. Furthermore, the upstream transcription factors NAC9 and NAC6 exhibited binding affinity toward the promoters of PheCLE1 and PheCLE10, thereby facilitating their transcriptional activation. In summary, this study not only systematically identified the CLE gene family in Moso bamboo for the first time but also emphasized their central roles in apical tissue development. This provides a valuable theoretical foundation for the further exploration of functional peptides and their signaling regulatory networks in bamboo species.

Deciphering the regulatory role of PheSnRK genes in Moso bamboo: insights into hormonal, energy, and stress responses.

The SnRK (sucrose non-fermentation-related protein kinase) plays an important role in regulating various signals in plants. However, as an important bamboo shoot and wood species, the response mechanism of PheSnRK in Phyllostachys edulis to hormones, low energy and stress remains unclear. In this paper, we focused on the structure, expression, and response of SnRK to hormones and sugars. In this study, we identified 75 PheSnRK genes from the Moso bamboo genome, which can be divided into three groups according to the evolutionary relationship. Cis-element analysis has shown that the PheSnRK gene can respond to various hormones, light, and stress. The PheSnRK2.9 proteins were localized in the nucleus and cytoplasm. Transgenic experiments showed that overexpression of PheSnRK2.9 inhibited root development, the plants were salt-tolerant and exhibited slowed starch consumption in Arabidopsis in the dark. The results of yeast one-hybrid and dual luciferase assay showed that PheIAAs and PheNACs can regulate PheSnRK2.9 gene expression by binding to the promoter of PheSnRK2.9. This study provided a comprehensive understanding of PheSnRK genes of Moso bamboo, which provides valuable information for further research on energy regulation mechanism and stress response during the growth and development of Moso bamboo.

Single-cell transcriptome atlas reveals spatiotemporal developmental trajectories in the basal roots of moso bamboo (Phyllostachys edulis).

Roots are essential for plant growth and development. Bamboo is a large Poaceae perennial with 1642 species worldwide. However, little is known about the transcriptional atlas that underpins root cell-type differentiation. Here, we set up a modified protocol for protoplast preparation and report single-cell transcriptomes of 14 279 filtered single cells derived from the basal root tips of moso bamboo. We identified four cell types and defined new cell-type-specific marker genes for the basal root. We reconstructed the developmental trajectories of the root cap, epidermis, and ground tissues and elucidated critical factors regulating cell fate determination. According to in situ hybridization and pseudotime trajectory analysis, the root cap and epidermis originated from a common initial cell lineage, revealing the particularity of bamboo basal root development. We further identified key regulatory factors for the differentiation of these cells and indicated divergent root developmental pathways between moso bamboo and rice. Additionally, PheWOX13a and PheWOX13b ectopically expressed in Arabidopsis inhibited primary root and lateral root growth and regulated the growth and development of the root cap, which was different from WOX13 orthologs in Arabidopsis. Taken together, our results offer an important resource for investigating the mechanism of root cell differentiation and root system architecture in perennial woody species of Bambusoideae.

Plasticity in the Morphology of Growing Bamboo: A Bayesian Analysis of Exogenous Treatment Effects on Plant Height, Internode Length, and Internode Numbers.

Sucrose (Suc) and gibberellin (GA) can promote the elongation of certain internodes in bamboo. However, there is a lack of field studies to support these findings and no evidence concerning how Suc and GA promote the plant height of bamboo by regulating the internode elongation and number. We investigated the plant height, the length of each internode, and the total number of internodes of Moso bamboo (Phyllostachys edulis) under exogenous Suc, GA, and control group (CTRL) treatments in the field and analyzed how Suc and GA affected the height of Moso bamboo by promoting the internode length and number. The lengths of the 10th-50th internodes were significantly increased under the exogenous Suc and GA treatments, and the number of internodes was significantly increased by the exogenous Suc treatment. The increased effect of Suc and GA exogenous treatment on the proportion of longer internodes showed a weakening trend near the plant height of 15-16 m compared with the CTRL, suggesting that these exogenous treatments may be more effective in regions where bamboo growth is suboptimal. This study demonstrated that both the exogenous Suc and GA treatments could promote internode elongation of Moso bamboo in the field. The exogenous GA treatment had a stronger effect on internode elongation, and the exogenous Suc treatment had a stronger effect on increasing the internode numbers. The increase in plant height by the exogenous Suc and GA treatments was promoted by the co-elongation of most internodes or the increase in the proportion of longer internodes.

PheGRF4e initiated auxin signaling during moso bamboo shoot development.

BACKGROUND: As a ubiquitous acid-regulating protein family in eukaryotes, general regulatory factors (GRFs) are active in various life activities of plants. However, detailed investigations of the GRFs gene family in moso bamboo are scarce. METHODS AND RESULTS: Genome-wide characteristics of the GRF gene family in moso bamboo were analyzed using the moso bamboo genome. GRF phylogeny, gene structure, conserved domains, cis-element promoters, and gene expression were systematically analyzed. A total of 20 GRF gene family members were identified in the moso bamboo genome. These genes were divided into ε and non-ε groups. qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction) showed that PheGRF genes responded to auxin and gibberellin treatment. To further study PheGRF gene functions, a yeast two-hybrid experiment was performed and verified by a bimolecular fluorescence complementation experiment. The results showed that PheGRF4e could interact with PheIAA30 (auxin/indole-3-acetic acid, an Aux/IAA family gene), and both were found to act mainly on the root tip meristem and vascular bundle cells of developing shoots by in situ hybridization assay. CONCLUSIONS: This study revealed that PheGRF genes were involved in hormone response during moso bamboo shoot development, and the possible regulatory functions of PheGRF genes were enriched by the fact that PheGRF4e initiated auxin signaling by binding to PheIAA30.

New Insights Into the Local Auxin Biosynthesis and Its Effects on the Rapid Growth of Moso Bamboo (Phyllostachys edulis).

Auxin plays a crucial regulatory role in higher plants, but systematic studies on the location of auxin local biosynthesis are rare in bamboo and other graminaceous plants. We studied moso bamboo (Phyllostachys edulis), which can grow up to 1 m/day and serves as a reference species for bamboo and other fast-growing species. We selected young tissues such as root tips, shoot tips, young culm sheaths, sheath blades, and internode divisions for local auxin biosynthesis site analysis. IAA immunofluorescence localization revealed that auxin was similarly distributed in different stages of 50-cm and 300-cm bamboo shoots. Shoot tips had the highest auxin content, and it may be the main site of auxin biosynthesis in the early stage of rapid growth. A total of 22 key genes in the YUCCA family for auxin biosynthesis were identified by genome-wide identification, and these had obvious tissue-specific and spatio-temporal expression patterns. In situ hybridization analysis revealed that the localization of YUCCA genes was highly consistent with the distribution of auxin. Six major auxin synthesis genes, PheYUC3-1, PheYUC6-1, PheYUC6-3, PheYUC9-1, PheYUC9-2, and PheYUC7-3, were obtained that may have regulatory roles in auxin accumulation during moso bamboo growth. Culm sheaths were found to serve as the main local sites of auxin biosynthesis and the auxin required for internode elongation may be achieved mainly by auxin transport.

Non-destructive detection of blueberry skin pigments and intrinsic fruit qualities based on deep learning.

BACKGROUND: This paper proposes a novel method to improve accuracy and efficiency in detecting the quality of blueberry fruit, taking advantage of deep learning in classification tasks. We first collected 'Tifblue' blueberries at seven different stages of maturity (10-70 days after full bloom) and measured the pigments of the blueberry skin and the total sugar and the total acid of the pulp. We then established a skin pigment contents prediction network (SPCPN), based on the correlation between the pigments and blueberry pictures, and also a fruit intrinsic qualities prediction network (FIQPN), based on the correlation between the pigments and fruit qualities. Finally, the SPCPN and FIQPN were consolidated into the blueberry quality parameters prediction network (BQPPN). RESULTS: The results showed that the anthocyanins in the blueberry skin were significantly correlated with the total sugar, total acid, and sugar / acid ratio of the fruit. After verification, the results also indicated that, for the prediction of anthocyanins, chlorophyll, and the anthocyanin / chlorophyll ratio, the SPCPN network model was found to achieve higher R2 (RMSE) values of 0.969 (0.139), 0.955 (0.005), 0.967 (15.4), respectively. The FIQPN network model was also able to evaluate the value of total sugar (R2 = 0.940, RMSE = 4.905), total acid (R2 = 0.930, RMSE = 2.034), and the sugar / acid ratio (R2 = 0.973, RMSE = 0.580). CONCLUSION: The above results indicated the potential for utilizing deep learning technology to predict the quality indicators of blueberry before harvesting. © 2020 Society of Chemical Industry.