Genome-wide identification of glycoside hydrolase family 1 members reveals GeBGL1 and GeBGL9 for degrading gastrodin in Gastrodia elata.

KEY MESSAGE: We revealed the intrinsic transformation molecular mechanism of gastrodin by two ß-d-glucosidases (GeBGL1 and GeBGL9) during the processing of Gastrodia elata. Gastrodia elata is a plant resource with medicinal and edible functions, and its active ingredient is gastrodin. However, the intrinsic transformation molecular mechanism of gastrodin in G. elata has not been verified. We speculated that ß-d-glucosidase (BGL) may be the key enzymes hydrolyzing gastrodin. Here, we identified 11 GeBGL genes in the G. elata genome. These genes were unevenly distributed on seven chromosomes. These GeBGL proteins possessed motifs necessary for catalysis, namely, TF(I/M/L)N(T)E(Q)P and I(V/L)T(H/S)ENG(S). These GeBGLs were divided into five subgroups together with homologous genes from Arabidopsis thaliana, rice, and maize. Quantitative real-time PCR analysis showed GeBGL genes expression was tissue-specific. Gene cloning results showed two mutation sites in the GeBGL1 gene compared with the reference genome. And, the GeBGL4 gene has two indel fragments, which resulted in premature termination of translation and seemed to turn into a pseudogene. Furthermore, protein expression and enzyme activity results proved that GeBGL1 and GeBGL9 have the activity of hydrolyzing gastrodin into 4-hydroxybenzyl alcohol. This study revealed the function of ß-d-glucosidase in degrading active compounds during the G. elata processing for medicinal purposes. These results offer a theoretical foundation for elevating the standard and enhancing the quality of G. elata production.

Genome-Wide Identification of the CYP716 Gene Family in Platycodon grandiflorus (Jacq.) A. DC. and Its Role in the Regulation of Triterpenoid Saponin Biosynthesis.

The main type of saponins occurring in the root of Platycodon grandiflorus (Jacq.) A. DC. are oleanolic acid glycosides. The CYP716 gene family plays a major role in catalyzing the conversion of ß-amyrin into oleanolic acid. However, studies on the CYP716 genes in P. grandiflorus are limited, and its evolutionary history remains poorly understood. In this study, 22 PgCYP716 genes were identified, distributed among seven subfamilies. Cis-acting elements of the PgCYP716 promoters were mainly involved in plant hormone regulation and responses to abiotic stresses. PgCYP716A264, PgCYP716A391, PgCYP716A291, and PgCYP716BWv3 genes were upregulated in the root and during saponin accumulation, as shown by RNA-seq analysis, suggesting that these four genes play an important role in saponin synthesis. The results of subcellular localization indicated that these four genes encoded membrane proteins. Furthermore, the catalytic activity of these four genes was proved in the yeast, which catalyzed the conversion of ß-amyrin into oleanolic acid. We found that the content of ß-amyrin, platycodin D, platycoside E, platycodin D3, and total saponins increased significantly when either of the four genes was over expressed in the transgenic hair root. In addition, the expression of PgSS, PgGPPS2, PgHMGS, and PgSE was also upregulated while these four genes were overexpressed. These data support that these four PgCYP716 enzymes oxidize ß-amyrin to produce oleanolic acid, ultimately promoting saponin accumulation by activating the expression of upstream pathway genes. Our results enhanced the understanding of the functional variation among the PgCYP716 gene family involved in triterpenoid biosynthesis and provided a theoretical foundation for improving saponin content and enriching the saponin biosynthetic pathway in P. grandiflorus.

Genome-wide identification and analysis of the cotton ALDH gene family.

BACKGROUND: Aldehyde dehydrogenases (ALDHs) are a family of enzymes that catalyze the oxidation of aldehyde molecules into the corresponding carboxylic acid, regulate the balance of aldehydes and protect plants from the poisoning caused by excessive accumulation of aldehydes; however, this gene family has rarely been studied in cotton. RESULTS: In the present study, genome-wide identification was performed, and a total of 114 ALDH family members were found in three cotton species, Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii. The ALDH genes were divided into six subgroups by evolutionary analysis. ALDH genes in the same subgroup showed similar gene structures and conserved motifs, but some genes showed significant differences, which may result in functional differences. Chromosomal location analysis and selective pressure analysis revealed that the ALDH gene family had experienced many fragment duplication events. Cis-acting element analysis revealed that this gene family may be involved in the response to various biotic and abiotic stresses. The RT‒qPCR results showed that the expression levels of some members of this gene family were significantly increased under salt stress conditions. Gohir.A11G040800 and Gohir.D06G046200 were subjected to virus-induced gene silencing (VIGS) experiments, and the sensitivity of the silenced plants to salt stress was significantly greater than that of the negative control plants, suggesting that Gohir.A11G040800 and Gohir.D06G046200 may be involved in the response of cotton to salt stress. CONCLUSIONS: In total, 114 ALDH genes were identified in three Gossypium species by a series of bioinformatics analysis. Gene silencing of the ALDH genes of G. hirsutum revealed that ALDH plays an important role in the response of cotton to salt stress.

[Identification and expression profile analysis of rice CRF gene family].

Cytokinin response factors (CRFs), as unique transcription factors in plants, play crucial roles in regulating development, phytohormone signaling pathway, and stress responses. In this study, we identified nine CRF genes from the rice genome by conducting a BLAST analysis using the protein sequences of twelve Arabidopsis AtCRFs. These genes are located on seven different rice chromosomes. We conducted a comprehensive analysis of the conserved domains, physicochemical properties, secondary structures, and phylogenetic relationships of rice CRF proteins using various online tools and local software. Additionally, we analyzed the exon-intron structures and cis-acting elements of OsCRFs, and found an abundance of elements relevant to phytohormone response and stress response on the promoters of rice CRF genes. Spatial-temporal expression pattern analysis revealed that four of the OsCRFs were barely expressed in all tested samples, while the other five were highly expressed in the leaf, panicle, or seed of rice. Microarray data showed that OsCRF genes are regulated to varying degrees by abscisic acid, auxin, cytokinin, and jasmonic acid. Furthermore, through analyzing the RNA-seq data, we found that OsCRFs are primarily involved in plant response to temperature stress (chilling and heat), with several OsCRFs also implicated in drought response, while hardly any respond to salt stress. This study provides an important basis for the functional characterization of rice CRF family genes.

Genome-wide identification of polyamine metabolism and ethylene synthesis genes in Chenopodium quinoa Willd. and their responses to low-temperature stress.

BACKGROUND: Quinoa (Chenopodium quinoa Willd.) is valued for its nutritional richness. However, pre-harvest sprouting poses a significant threat to yield and grain quality. This study aims to enhance our understanding of pre-harvest sprouting mitigation strategies, specifically through delayed sowing and avoiding rainy seasons during quinoa maturation. The overarching goal is to identify cold-resistant varieties and unravel the molecular mechanisms behind the low-temperature response of quinoa. We employed bioinformatics and genomics tools for a comprehensive genome-wide analysis of polyamines (PAs) and ethylene synthesis gene families in quinoa under low-temperature stress. RESULTS: This involved the identification of 37 PA biosynthesis and 30 PA catabolism genes, alongside 227 ethylene synthesis. Structural and phylogenetic analyses showcased conserved patterns, and subcellular localization predictions indicated diverse cellular distributions. The results indicate that the PA metabolism of quinoa is closely linked to ethylene synthesis, with multiple genes showing an upregulation in response to cold stress. However, differential expression within gene families suggests a nuanced regulatory network. CONCLUSIONS: Overall, this study contributes valuable insights for the functional characterization of the PA metabolism and ethylene synthesis of quinoa, which emphasize their roles in plant low-temperature tolerance and providing a foundation for future research in this domain.

MADS-Box Family Genes in Lagerstroemia indica and Their Involvement in Flower Development.

MADS-box is a key transcription factor regulating the transition to flowering and flower development. Lagerstroemia indica 'Xiang Yun' is a new cultivar of crape myrtle characterized by its non-fruiting nature. To study the molecular mechanism underlying the non-fruiting characteristics of 'Xiang Yun', 82 MADS-box genes were identified from the genome of L. indica. The physicochemical properties of these genes were examined using bioinformatics methods, and their expression as well as endogenous hormone levels at various stages of flower development were analyzed. The results showed that LiMADS genes were primarily classified into two types: type I and type II, with the majority being type II that contained an abundance of cis-acting elements in their promoters. By screening nine core proteins by predicted protein interactions and performing qRT-PCR analysis as well as in combination with transcriptome data, we found that the expression levels of most MADS genes involved in flower development were significantly lower in 'Xiang Yun' than in the wild type 'Hong Ye'. Hormonal analysis indicated that 'Xiang Yun' had higher levels of iP, IPR, TZR, and zeatin during its early stages of flower development than 'Hong Ye', whereas the MeJA content was substantially lower at the late stage of flower development of 'Hong Ye'. Finally, correlation analysis showed that JA, IAA, SA, and TZR were positively correlated with the expression levels of most type II genes. Based on these analyses, a working model for the non-fruiting 'Xiang Yun' was proposed. During the course of flower development, plant hormone response pathways may affect the expression of MADS genes, resulting in their low expression in flower development, which led to the abnormal development of the stamen and embryo sac and ultimately affected the fruiting process of 'Xiang Yun'.

Genome-Wide Analysis of the DC1 Domain Protein Gene Family in Tomatoes under Abiotic Stress.

DC1 (Divergent C1) domain proteins are a new class of proteins that have been discovered in recent years, which play an important role in plant growth, development, and stress response. In order to better study the distribution and function of DC1 domain proteins in tomatoes, a genome-wide identification was conducted. It was found that there are twenty-one DC1 domain protein genes distributed on nine chromosomes of tomatoes, named SlCHP1-21. Phylogenetic analysis shows that twenty-one SlCHP genes are divided into six subfamilies. Most of the SlCHP genes in tomatoes have no or very short introns. All SlCHP proteins, with the exception of SlCHP8 and SlCHP17, contain variable amounts of C1 domain. Analysis of the SlCHP gene promoter sequence revealed multiple cis-elements responsive to plant stress. qRT-CR analysis showed that most members of SlCHP gene expressed in the roots. The SlCHP11, 13, 16, 17, and SlCHP20 genes showed specific responses to high temperature, low temperature, salt, and drought stress. In addition, the subcellular localization and interaction proteins of SlCHP were analyzed and predicted. Together, these results provides a theoretical basis for further exploration of the function and mechanism of the SlCHP gene in tomatoes.

Genome-Wide Identification of Callose Synthase Family Genes and Their Expression Analysis in Floral Bud Development and Hormonal Responses in Prunus mume.

Callose is an important polysaccharide composed of beta-1,3-glucans and is widely implicated in plant development and defense responses. Callose synthesis is mainly catalyzed by a family of callose synthases, also known as glucan synthase-like (GSL) enzymes. Despite the fact that GSL family genes were studied in a few plant species, their functional roles have not been fully understood in woody perennials. In this study, we identified total of 84 GSL genes in seven plant species and classified them into six phylogenetic clades. An evolutionary analysis revealed different modes of duplication driving the expansion of GSL family genes in monocot and dicot species, with strong purifying selection constraining the protein evolution. We further examined the gene structure, protein sequences, and physiochemical properties of 11 GSL enzymes in Prunus mume and observed strong sequence conservation within the functional domain of PmGSL proteins. However, the exon-intron distribution and protein motif composition are less conservative among PmGSL genes. With a promoter analysis, we detected abundant hormonal responsive cis-acting elements and we inferred the putative transcription factors regulating PmGSLs. To further understand the function of GSL family genes, we analyzed their expression patterns across different tissues, and during the process of floral bud development, pathogen infection, and hormonal responses in Prunus species and identified multiple GSL gene members possibly implicated in the callose deposition associated with bud dormancy cycling, pathogen infection, and hormone signaling. In summary, our study provides a comprehensive understanding of GSL family genes in Prunus species and has laid the foundation for future functional research of callose synthase genes in perennial trees.

Genome-Wide Identification and Expression Patterns of Cucumber Invertases and Their Inhibitor Genes.

Invertases and their inhibitors play important roles in sucrose metabolism, growth and development, signal transduction, and biotic and abiotic stress tolerance in many plant species. However, in cucumber, both the gene members and functions of invertase and its inhibitor families remain largely unclear. In this study, in comparison with the orthologues of Citrullus lanatus (watermelon), Cucumis melo (melon), and Arabidopsis thaliana (Arabidopsis), 12 invertase genes and 12 invertase inhibitor genes were identified from the genome of Cucumis sativus (cucumber). Among them, the 12 invertase genes were classified as 4 cell wall invertases, 6 cytoplasmic invertases, and 2 vacuolar invertases. Most invertase genes were conserved in cucumber, melon, and watermelon, with several duplicate genes in melon and watermelon. Transcriptome analysis distinguished these genes into various expression patterns, which included genes CsaV3_2G025540 and CsaV3_2G007220, which were significantly expressed in different tissues, organs, and development stages, and genes CsaV3_7G034730 and CsaV3_5G005910, which might be involved in biotic and abiotic stress. Six genes were further validated in cucumber based on quantitative real-time PCR (qRT-PCR), and three of them showed consistent expression patterns as revealed in the transcriptome. These results provide important information for further studies on the physiological functions of cucumber invertases (CSINVs) and their inhibitors (CSINHs).

Exploring the role of detoxification genes in the resistance of Bursaphelenchus xylophilus to different exogenous nematicidal substances using transcriptomic analyses.

Bursaphelenchus xylophilus (Pine wood nematode, PWN) has become a worldwide forest disease due to its rapid infection ability, high lethality and difficulty in control. The main means of countering B. xylophilus is currently chemical control, but nematicides can present problems such as environmental pollution and drug resistance. The development of novel environmentally-friendly nematicides has thus become a focus of recent research. In this study, BxUGT3 and BxUGT34, which might be related to detoxification, were investigated by comparing transcriptomic and WGCNA approaches. Three other genes with a similar expression pattern, BxUGT13, BxUGT14, and BxUGT16, were found by gene family analysis. Further bioassays and qPCR assays confirmed that these five genes showed significant changes in transcript levels upon exposure to α-pinene and carvone, demonstrating that they respond to exogenous nematicidal substances. Finally, RNAi and bioassays showed that B. xylophilus with silenced BxUGT16 had increased mortality in the face of α-pinene and carvone stress, suggesting that BxUGT16 plays an important role in detoxification. Taken together, this study used novel molecular research methods, explored the detoxification mechanism of B. xylophilus at a transcriptomic level, and revealed a molecular target for the development of novel biopesticides.

Genome-Wide Identification of PEBP Gene Family in Solanum lycopersicum.

The PEBP gene family is crucial for the growth and development of plants, the transition between vegetative and reproductive growth, the response to light, the production of florigen, and the reaction to several abiotic stressors. The PEBP gene family has been found in numerous species, but the SLPEBP gene family has not yet received a thorough bioinformatics investigation, and the members of this gene family are currently unknown. In this study, bioinformatics was used to identify 12 members of the SLPEBP gene family in tomato and localize them on the chromosomes. The physicochemical characteristics of the proteins encoded by members of the SLPEBP gene family were also examined, along with their intraspecific collinearity, gene structure, conserved motifs, and cis-acting elements. In parallel, a phylogenetic tree was built and the collinear relationships of the PEBP gene family among tomato, potato, pepper, and Arabidopsis were examined. The expression of 12 genes in different tissues and organs of tomato was analyzed using transcriptomic data. It was also hypothesized that SLPEBP3, SLPEBP5, SLPEBP6, SLPEBP8, SLPEBP9, and SLPEBP10 might be related to tomato flowering and that SLPEBP2, SLPEBP3, SLPEBP7, and SLPEBP11 might be related to ovary development based on the tissue-specific expression analysis of SLPEBP gene family members at five different stages during flower bud formation to fruit set. This article's goal is to offer suggestions and research directions for further study of tomato PEBP gene family members.

Identification of ABF/AREB gene family in tomato (Solanum lycopersicum L.) and functional analysis of ABF/AREB in response to ABA and abiotic stresses.

Abscisic acid (ABA) is a plant hormone that plays an important regulatory role in plant growth and stress response. The AREB (ABA-responsive element binding protein)/ABF (ABRE-binding factor) are important ABA-signaling components that participate in abiotic stress response. However, genome-scale analysis of ABF/AREB has not been systemically investigated in tomato. This study was conducted to identify tomato ABF/AREB family members and analyze their response to ABA and abiotic stresses. The results show that a total of 10 ABF/AREB members were identified in tomato, which are randomly distributed on five chromosomes. Domain analysis showed that these members exhibit high protein similarity, especially in the basic leucine zipper (bZIP) domain region. Subcellular localization analysis indicated that all 10 ABF/AREB members are localized in the nucleus. Phylogenetic tree analysis showed that tomato ABF/AREB genes are divided into two groups, and they are similar with the orthologs of other plants. The analysis of cis-acting elements showed that most tomato ABF/AREB genes contain a variety of hormones and stress-related elements. Expression profiles of different tissues indicated that SlABF2 and SlABF10 play an important role in fruit ripening. Finally, qRT-PCR analysis revealed that 10 tomato ABF/AREB genes respond to ABA, with SlABF3 being the most sensitive. SlABF3, SlABF5 and SlABF10 positively respond to salt and cold stresses. SlABF1, SlABF3 and SlABF10 are significantly induced under UV radiation treatment. SlABF3 and SlABF5 are significantly induced in osmotic stress. Overall, this study may provide insight into the role of tomato ABF/AREB homologues in plant response to abiotic stresses, which laid a foundation for future functional study of ABF/AREB in tomato.

Genome-Wide Re-Identification and Analysis of CrRLK1Ls in Tomato.

The Catharanthus roseus receptor-like kinase 1-like (CrRLK1L), which is a vital member of the plant receptor-like kinase family, plays versatile roles in plant growth, development, and stress response. Although the primary screening of tomato CrRLK1Ls has been reported previously, our knowledge of these proteins is still scarce. Using the latest genomic data annotations, a genome-wide re-identification and analysis of the CrRLK1Ls in tomatoes were conducted. In this study, 24 CrRLK1L members were identified in tomatoes and researched further. Subsequent gene structures, protein domains, Western blot analyses, and subcellular localization analyses all confirmed the accuracy of the newly identified SlCrRLK1L members. Phylogenetic analyses showed that the identified SlCrRLK1L proteins had homologs in Arabidopsis. Evolutionary analysis indicated that two pairs of the SlCrRLK1L genes had predicted segmental duplication events. Expression profiling analyses demonstrated that the SlCrRLK1L genes were expressed in various tissues, and most of them were up- or down-regulated by bacteria and PAMP treatments. Together, these results will lay the foundation for elaborating the biological roles of SlCrRLK1Ls in tomato growth, development, and stress response.

Genome-Wide Identification, Evolution, and Expression Analysis of GASA Gene Family in Prunus mume.

The Gibberellic Acid Stimulated Arabidopsis/Gibberellin Stimulated Transcript (GASA/GAST) gene family is a group of plant-specific genes encoding cysteine-rich peptides essential to plant growth, development, and stress responses. Although GASA family genes have been identified in various plant species, their functional roles in Prunus mume are still unknown. In this study, a total of 16 PmGASA genes were identified via a genome-wide scan in Prunus mume and were grouped into three major gene clades based on the phylogenetic tree. All PmGASA proteins possessed the conserved GASA domain, consisting of 12-cysteine residues, but varied slightly in protein physiochemical properties and motif composition. With evolutionary analysis, we observed that duplications and purifying selection are major forces driving PmGASA family gene evolution. By analyzing PmGASA promoters, we detected a number of hormonal-response related cis-elements and constructed a putative transcriptional regulatory network for PmGASAs. To further understand the functional role of PmGASA genes, we analyzed the expression patterns of PmGASAs across different organs and during various biological processes. The expression analysis revealed the functional implication of PmGASA gene members in gibberellic acid-, abscisic acid-, and auxin-signaling, and during the progression of floral bud break in P. mume. To summarize, these findings provide a comprehensive understanding of GASA family genes in P. mume and offer a theoretical basis for future research on the functional characterization of GASA genes in other woody perennials.

Genome-wide identification and analysis of ACP gene family in Sorghum bicolor (L.) Moench.

BACKGROUND: Acyl carrier proteins (ACP) constitute a very conserved carrier protein family. Previous studies have found that ACP not only takes part in the fatty acid synthesis process of almost all organisms, but also participates in the regulation of plant growth, development, and metabolism, and makes plants adaptable to stresses. However, this gene family has not been systematically studied in sorghum. RESULTS: Nine ACP family members were identified in the sorghum genome, which were located on chromosomes 1, 2, 5, 7, 8 and 9, respectively. Evolutionary analysis among different species divided the ACP family into four subfamilies, showing that the SbACPs were more closely related to maize. The prediction results of subcellular localization showed that SbACPs were mainly distributed in chloroplasts and mitochondria, while fluorescence localization showed that SbACPs were mainly localized in chloroplasts in tobacco leaf. The analysis of gene structure revealed a relatively simple genetic structure, that there were 1-3 introns in the sorghum ACP family, and the gene structure within the same subfamily had high similarity. The amplification method of SbACPs was mainly large fragment replication, and SbACPs were more closely related to ACPs in maize and rice. In addition, three-dimensional structure analysis showed that all ACP genes in sorghum contained four α helices, and the second helix structure was more conserved, implying a key role in function. Cis-acting element analysis indicated that the SbACPs might be involved in light response, plant growth and development regulation, biotic and abiotic stress response, plant hormone regulation, and other physiological processes. What's more, qRT-PCR analysis uncovered that some of SbACPs might be involved in the adaptive regulation of drought and salt stresses, indicating the close relationship between fatty acids and the resistance to abiotic stresses in sorghum. CONCLUSIONS: In summary, these results showed a comprehensive overview of the SbACPs and provided a theoretical basis for further studies on the biological functions of SbACPs in sorghum growth, development and abiotic stress responses.

The Identification of Broomcorn Millet bZIP Transcription Factors, Which Regulate Growth and Development to Enhance Stress Tolerance and Seed Germination.

Broomcorn millet (Panicum miliaceum L.) is a water-efficient and highly salt-tolerant plant. In this study, the salt tolerance of 17 local species of broomcorn millet was evaluated through testing based on the analysis of the whitening time and the germination rate of their seeds. Transcriptome sequencing revealed that PmbZIP131, PmbZIP125, PmbZIP33, PmABI5, PmbZIP118, and PmbZIP97 are involved in seed germination under salt stress. Seedling stage expression analysis indicates that PmABI5 expression was induced by treatments of high salt (200 mM NaCl), drought (20% W/V PEG6000), and low temperature (4 °C) in seedlings of the salt-tolerant variety Y9. The overexpression of PmABI5 significantly increases the germination rate and root traits of Arabidopsis thaliana transgenic lines, with root growth and grain traits significantly enhanced compared to the wild type (Nipponbare). BiFC showed that PmABI5 undergoes homologous dimerization in addition to forming a heterodimer with either PmbZIP33 or PmbZIP131. Further yeast one-hybrid experiments showed that PmABI5 and PmbZIP131 regulate the expression of PmNAC1 by binding to the G-box in the promoter. These results indicate that PmABI5 can directly regulate seed germination and seedling growth and indirectly improve the salt tolerance of plants by regulating the expression of the PmNAC1 gene through the formation of heterodimers with PmbZIP131.

Genome-wide identification of PEBP gene family members in potato, their phylogenetic relationships, and expression patterns under heat stress.

BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP) gene family is involved in regulating many plant traits. Genome-wide identification of PEPB members and knowledge of their responses to heat stress may assist genetic improvement of potato (Solanum tuberosum). METHODS AND RESULTS: We identified PEBP gene family members from both the recently-updated, long-reads-based reference genome (DM v6.1) and the previous short-reads-based annotation (PGSC DM v3.4) of the potato reference genome and characterized their heat-induced gene expression using RT-PCR and RNA-Seq. Fifteen PEBP family genes were identified from DM v6.1 and named as StPEBP1 to StPEBP15 based on their locations on 6 chromosomes and were classified into FT, TFL, MFT, and PEBP-like subfamilies. Most of the StPEBP genes were found to have conserved motifs 1 to 5. Tandem or segmental duplications were found between StPEBP genes in seven pairs. Heat stress induced opposite expression patterns of certain FT and TFL members but involving different members in leaves, roots and tubers. CONCLUSION: The long-reads-based genome assembly and annotation provides a better genomic resource for identification of PEBP family genes. Heat stress tends to decrease FT gene activities but increases TFL gene activities, but this opposite expression involves different FT/TFL pairs in leaves, roots, and tubers. This tissue-specific expression pattern of PEBP members may partly explain why different potato organs differ in their sensitivities to heat stress. Our study provides candidate PEBP family genes and relevant information for genetic improvement of heat tolerance in potato and may help understand heat-induced responses in other plants.

Genome-Wide Identification of Maize Aquaporin and Functional Analysis During Seed Germination and Seedling Establishment.

Water uptake facilitates the initiation of seed germination. It is presumed that aquaporin (AQP)-mediated water inflow contributes to seed germination, but the genetic evidence is still lacking. This study aimed at genome-wide identification of ZmAQPs and further determined the physiological functions. Following a comprehensive search, a total of 41 ZmAQPs were identified according to the latest genome database. Through bioinformatic approaches, the physicochemical characteristics, phylogenetic relationships, and structural features of ZmAQPs were analyzed. The gene expression analysis of 20 high-resolution and multi-tissues samples showed that ZmAQPs had distinct spatiotemporal and tissue-specific expression profiles during seed germination and early seedling development. We then focused on the aquaporin of maize tonoplast intrinsic protein 3 (ZmTIP3), which is specifically expressed in germinating seed. A mutant zmtip3-1 with disruption of the ZmTIP3-1 gene showed shorter shoot and root length, and decreased seedling dry weight compared with the control (W22). The result revealed that ZmTIP3-1 improved the absolute content of seed protein and promoted storage reserves mobilization, suggesting that ZmTIP3 may be a positive regulator of seed vigor. This work provides valuable clues for understanding the function and possible regulatory mechanism of ZmAQPs in seed germination and seedling growth.

Genome-wide analysis of the Populus trichocarpa laccase gene family and functional identification of PtrLAC23.

Introduction: Biofuel is a kind of sustainable, renewable and environment friendly energy. Lignocellulose from the stems of woody plants is the main raw material for "second generation biofuels". Lignin content limits fermentation yield and is therefore a major obstacle in biofuel production. Plant laccase plays an important role in the final step of lignin formation, which provides a new strategy for us to obtain ideal biofuels by regulating the expression of laccase genes to directly gain the desired lignin content or change the composition of lignin. Methods: Multiple sequence alignment and phylogenetic analysis were used to classify PtrLAC genes; sequence features of PtrLACs were revealed by gene structure and motif composition analysis; gene duplication, interspecific collinearity and Ka/Ks analysis were conducted to identify ancient PtrLACs; expression levels of PtrLAC genes were measured by RNA-Seq data and qRT-PCR; domain analysis combine with cis-acting elements prediction together showed the potential function of PtrLACs. Furthermore, Alphafold2 was used to simulate laccase 3D structures, proLAC23::LAC23-eGFP transgenic Populus stem transects were applied to fluorescence observation. Results: A comprehensive analysis of the P. trichocarpa laccase gene (PtLAC) family was performed. Some ancient PtrLAC genes such as PtrLAC25, PtrLAC19 and PtrLAC41 were identified. Gene structure and distribution of conserved motifs clearly showed sequence characteristics of each PtrLAC. Combining published RNA-Seq data and qRT-PCR analysis, we revealed the expression pattern of PtrLAC gene family. Prediction results of cis-acting elements show that PtrLAC gene regulation was closely related to light. Through above analyses, we selected 5 laccases and used Alphafold2 to simulate protein 3D structures, results showed that PtrLAC23 may be closely related to the lignification. Fluorescence observation of proLAC23::LAC23-eGFP transgenic Populus stem transects and qRT-PCR results confirmed our hypothesis again. Discussion: In this study, we fully analyzed the Populus trichocarpa laccase gene family and identified key laccase genes related to lignification. These findings not only provide new insights into the characteristics and functions of Populus laccase, but also give a new understanding of the broad prospects of plant laccase in lignocellulosic biofuel production.