Genome-wide identification and expression analysis of GRAS gene family in Eucalyptus grandis.

BACKGROUND: The GRAS gene family is a class of plant-specific transcription factors with important roles in many biological processes, such as signal transduction, disease resistance and stress tolerance, plant growth and development. So far, no information available describes the functions of the GRAS genes in Eucalyptus grandis. RESULTS: A total of 82 GRAS genes were identified with amino acid lengths ranging from 267 to 817 aa, and most EgrGRAS genes had one exon. Members of the GRAS gene family of Eucalyptus grandis are divided into 9 subfamilies with different protein structures, while members of the same subfamily have similar gene structures and conserved motifs. Moreover, these EgrGRAS genes expanded primarily due to segmental duplication. In addition, cis-acting element analysis showed that this family of genes was involved involved in the signal transduction of various plant hormones, growth and development, and stress response. The qRT-PCR data indicated that 18 EgrGRAS genes significantly responded to hormonal and abiotic stresses. Among them, the expression of EgrGRAS13, EgrGRAS68 and EgrGRAS55 genes was significantly up-regulated during the treatment period, and it was hypothesised that members of the EgrGRAS family play an important role in stress tolerance. CONCLUSIONS: In this study, the phylogenetic relationship, conserved domains, cis-elements and expression patterns of GRAS gene family of Eucalyptus grandis were analyzed, which filled the gap in the identification of GRAS gene family of Eucalyptus grandis and laid the foundation for analyzing the function of EgrGRAS gene in hormone and stress response.

Genome-wide identification and expression analysis of the GRAS family under low-temperature stress in bananas.

Introduction: GRAS, named after GAI, RGA, and SCR, is a class of plant-specific transcription factors family that plays a crucial role in growth and development, signal transduction, and various stress responses. Methods: To understand the biological functions of the banana GRAS gene family, a genome-wide identification and bioinformatics analysis of the banana GRAS gene family was performed based on information from the M. acuminata, M. balbisiana, and M. itinerans genomic databases. Result: In the present study, we identified 73 MaGRAS, 59 MbGRAS, and 58 MiGRAS genes in bananas at the whole-genome scale, and 56 homologous genes were identified in the three banana genomes. Banana GRASs can be classified into 10 subfamilies, and their gene structures revealed that most banana GRAS gDNAs lack introns. The promoter sequences of GRASs had a large number of cis-acting elements related to plant growth and development, phytohormone, and adversity stress responsiveness. The expression pattern of seven key members of MaGRAS response to low-temperature stress and different tissues was also examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microRNAs-MaGRASs target prediction showed perfect complementarity of seven GRAS genes with the five mac-miRNAs. The expression of all seven genes was lowest in roots, and the expression of five genes was highest in leaves during low-temperature stress. The expression of MaSCL27-2, MaSCL27-3, and MaSCL6-1 was significantly lower under low-temperature stress compared to the control, except for MaSCL27-2, which was slightly higher than the 28°C control at 4 h. The expression of MaSCL27-2, MaSCL27-3, and MaSCL6-1 dropped to the lowest levels at 24 h, 12 h, and 4 h, respectively. The MaSCL27-4 and MaSCL6-2 expression was intermittently upregulated, rising to the highest expression at 24h, while the expression of MaSCL22 was less variable, remaining at the control level with small changes. Discussion: In summary, it is tentatively hypothesized that the GRAS family has an important function in low-temperature stress in bananas. This study provides a theoretical basis for further analyzing the function of the banana GRAS gene and the resistance of bananas to cold temperatures.

Properties of INDETERMINATE DOMAIN Proteins from Physcomitrium patens: DNA-Binding, Interaction with GRAS Proteins, and Transcriptional Activity.

INDETERMINATE DOMAIN (IDD) proteins are plant-specific transcription factors that interact with GRAS proteins, such as DELLA and SHORT ROOT (SHR), to regulate target genes. The combination of IDD and DELLA proteins regulates genes involved in gibberellic acid (GA) synthesis and GA signaling, whereas the combination of IDD with the complex of SHR and SCARECROW, another GRAS protein, regulates genes involved in root tissue formation. Previous bioinformatic research identified seven IDDs, two DELLA, and two SHR genes in Physcomitrium patens, a model organism for non-vascular plants (bryophytes), which lack a GA signaling pathway and roots. In this study, DNA-binding properties and protein-protein interaction of IDDs from P. patens (PpIDD) were analyzed. Our results showed that the DNA-binding properties of PpIDDs were largely conserved between moss and seed plants. Four PpIDDs showed interaction with Arabidopsis DELLA (AtDELLA) proteins but not with PpDELLAs, and one PpIDD showed interaction with PpSHR but not with AtSHR. Moreover, AtIDD10 (JACKDAW) interacted with PpSHR but not with PpDELLAs. Our results indicate that DELLA proteins have modified their structure to interact with IDD proteins during evolution from moss lineage to seed plants, whereas the interaction of IDD and SHR was already present in moss lineage.

MYB3R-SCL28-SMR module with a role in cell size control negatively regulates G2 progression in Arabidopsis.

Cell size control is one of the prerequisites for plant growth and development. Recently, a GRAS family transcription factor, SCARECROW-LIKE28 (SCL28), was identified as a critical regulator for both mitotic and postmitotic cell-size control. Here, we show that SCL28 is specifically expressed in proliferating cells and exerts its function to delay G2 progression during mitotic cell cycle in Arabidopsis thaliana. Overexpression of SCL28 provokes a significant enlargement of cells in various organs and tissues, such as leaves, flowers and seeds, to different extents depending on the type of cells. The increased cell size is most likely due to a delayed G2 progression and accelerated onset of endoreplication, an atypical cell cycle repeating DNA replication without cytokinesis or mitosis. Unlike DWARF AND LOW-TILLERING, a rice ortholog of SCL28, SCL28 may not have a role in brassinosteroid (BR) signaling because sensitivity against brassinazole, a BR biosynthesis inhibitor, was not dramatically altered in scl28 mutant and SCL28-overexpressing plants. Collectively, our findings strengthen a recently proposed model of cell size control by SCL28 and suggest the presence of diversified evolutionary mechanisms for the regulation and action of SCL28.

Genome-wide identification, evolution and transcriptome analysis of GRAS gene family in Chinese chestnut (Castanea mollissima).

GRAS transcription factors play an important role in regulating various biological processes in plant growth and development. However, their characterization and potential function are still vague in Chinese chestnut (Castanea mollissima), an important nut with rich nutrition and high economic value. In this study, 48 CmGRAS genes were identified in Chinese chestnut genome and phylogenetic analysis divided CmGRAS genes into nine subfamilies, and each of them has distinct conserved structure domain and features. Genomic organization revealed that CmGRAS tend to have a representative GRAS domain and fewer introns. Tandem duplication had the greatest contribution for the CmGRAS expansion based on the comparative genome analysis, and CmGRAS genes experienced strong purifying selection pressure based on the Ka/Ks. Gene expression analysis revealed some CmGRAS members with potential functions in bud development and ovule fertility. CmGRAS genes with more homologous relationships with reference species had more cis-acting elements and higher expression levels. Notably, the lack of DELLA domain in members of the DELLA subfamily may cause de functionalization, and the differences between the three-dimensional structures of them were exhibited. This comprehensive study provides theoretical and practical basis for future research on the evolution and function of GRAS gene family.

Genome-Wide Identification, Characterization, and Expression Analysis of GRAS Gene Family in Ginger (Zingiber officinale Roscoe).

GRAS family proteins are one of the most abundant transcription factors in plants; they play crucial roles in plant development, metabolism, and biotic- and abiotic-stress responses. The GRAS family has been identified and functionally characterized in some plant species. However, this family in ginger (Zingiber officinale Roscoe), a medicinal crop and non-prescription drug, remains unknown to date. In the present study, 66 GRAS genes were identified by searching the complete genome sequence of ginger. The GRAS family is divided into nine subfamilies based on the phylogenetic analyses. The GRAS genes are distributed unevenly across 11 chromosomes. By analyzing the gene structure and motif distribution of GRAS members in ginger, we found that the GRAS genes have more than one cis-acting element. Chromosomal location and duplication analysis indicated that whole-genome duplication, tandem duplication, and segmental duplication may be responsible for the expansion of the GRAS family in ginger. The expression levels of GRAS family genes are different in ginger roots and stems, indicating that these genes may have an impact on ginger development. In addition, the GRAS genes in ginger showed extensive expression patterns under different abiotic stresses, suggesting that they may play important roles in the stress response. Our study provides a comprehensive analysis of GRAS members in ginger for the first time, which will help to better explore the function of GRAS genes in the regulation of tissue development and response to stress in ginger.

N-terminal region is required for functions of the HAM family member.

Shoot meristems contain stem cells, and they sustain growth and development of the above-ground tissues in land plants. The HAIRY MERISTEM (HAM) family genes, encoding GRAS-domain transcriptional regulators, play essential roles in the control of shoot meristem development and stem cell homeostasis in several flowering plants. Similar to other GRAS proteins, the C-terminal regions of HAM family proteins across land plants are conserved, containing signature motifs that define the GRAS domain. In contrast, the N-terminal regions of HAM family proteins display substantial divergence in sequence and length. Whether the variable and divergent N-termini are required for the conserved functions of HAM proteins is unknown. Our recent work showed that CrHAM - the HAM homolog in the fern Ceratopteris richardii was able to replace the role of type-II HAM genes in Arabidopsis, maintaining established shoot apical meristems and promoting the initiation of new stem cell niches. Here, we provide additional information and show that CrHAM contains a much longer N-terminal region compared to Arabidopsis HAM proteins, which is conserved among different fern HAM homologs. The deletion of this region largely compromises the ability of CrHAM to replace the function of Arabidopsis HAM proteins in shoot meristems. These new data together with previous results suggest that, although lacking the sequence conservation among HAM homologs from different plant lineages, the N-termini are important for the conserved functions of HAM family proteins.

PpGRAS12 acts as a positive regulator of meristem formation in Physcomitrium patens.

KEY MESSAGE: This study focused on the key regulatory function of Physcomitrium patens GRAS12 gene underlying an increasing plant complexity, an important step in plant terrestrialization and the evolutionary history of life. The miR171-GRAS module has been identified as a key player in meristem maintenance in angiosperms. PpGRAS12 is a member of the GRAS family and a validated target for miR171 in Physcomitrium (Physcomitrella) patens. Here we show a regulatory function of miR171 at the gametophytic vegetative growth stage and targeted deletion of the PpGRAS12 gene adversely affects sporophyte production since fewer sporophytes were produced in ΔPpGRAS12 knockout lines compared to wild type moss. Furthermore, highly specific and distinct growth arrests were observed in inducible PpGRAS12 overexpression lines at the protonema stage. Prominent phenotypic aberrations including the formation of multiple apical meristems at the gametophytic vegetative stage in response to elevated PpGRAS12 transcript levels were discovered via scanning electron microscopy. The production of multiple buds in the PpGRAS12 overexpression lines similar to ΔPpCLV1a/1b disruption mutants is accompanied by an upregulation of PpCLE and downregulation of PpCLV1, PpAPB, PpNOG1, PpDEK1, PpRPK2 suggesting that PpGRAS12 acts upstream of these genes and negatively regulates the proposed pathway to specify simplex meristem formation. As CLV signaling pathway components are not present in the chlorophytic or charophytic algae and arose with the earliest land plants, we identified a key regulatory function of PpGRAS12 underlying an increasing plant complexity, an important step in plant terrestrialization and the evolutionary history of life.

Transcriptomic analysis reveals the GRAS family genes respond to gibberellin in Salvia miltiorrhiza hairy roots.

BACKGROUND: Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants with high medicinal value. Gibberellins are growth-promoting phytohormones that regulate numerous growth and developmental processes in plants. However, their role on the secondary metabolism regulation has not been investigated. RESULTS: In this study, we found that gibberellic acid (GA) can promote hairy roots growth and increase the contents of tanshinones and phenolic acids. Transcriptomic sequencing revealed that many genes involved in the secondary metabolism pathway were the GA-responsive. After further analysis of GA signaling pathway genes, which their expression profiles have significantly changed, it was found that the GRAS transcription factor family had a significant response to GA. We identified 35 SmGRAS genes in S. miltiorrhiza, which can be divided into 10 subfamilies. Thereafter, members of the same subfamily showed similar conserved motifs and gene structures, suggesting possible conserved functions. CONCLUSIONS: Most SmGRAS genes were significantly responsive to GA, indicating that they may play an important role in the GA signaling pathway, also participating in the GA regulation of root growth and secondary metabolism in S. miltiorrhiza.

Gene Regulation via the Combination of Transcription Factors in the INDETERMINATE DOMAIN and GRAS Families.

INDETERMINATE DOMAIN (IDD) family proteins are plant-specific transcription factors. Some Arabidopsis IDD (AtIDD) proteins regulate the expression of SCARECROW (SCR) by interacting with GRAS family transcription factors SHORT-ROOT (SHR) and SCR, which are involved in root tissue formation. Some AtIDD proteins regulate genes involved in the synthesis (GA3ox1) or signaling (SCL3) of gibberellic acid (GA) by interacting with DELLA proteins, a subfamily of the GRAS family. We analyzed the DNA binding properties and protein-protein interactions of select AtIDD proteins. We also investigated the transcriptional activity of the combination of AtIDD and GRAS proteins (AtIDD proteins combined with SHR and SCR or with REPRESSOR of ga1-3 (RGA)) on the promoters of SCR,SCL3, and GA3ox1 by conducting a transient assay using Arabidopsis culture cells. Our results showed that the SCR promoter could be activated by the IDD and RGA complexes and that the SCL3 and GA3ox1 promoters could be activated by the IDD, SHR, and SCR complexes, indicating the possibility that these complexes regulate and consequently coordinate the expression of genes involved in GA synthesis (GA3ox1), GA signaling (SCL3), and root formation (SCR).

Genome-wide survey and expression analyses of the GRAS gene family in Brassica napus reveals their roles in root development and stress response.

MAIN CONCLUSION: Genome-wide identification, classification, expression analyses, and functional characterization of GRAS genes in oil crop, Brassica napus, indicate their importance in root development and stress response. GRAS proteins are a plant-specific transcription factor gene family involved in tissues development and stress response. We classified 87 putative GRAS genes in the Brassica napus genome (BnGRASs) into 13 subfamilies by phylogenetic analysis. The C-terminal GRAS domains of Brassica napus (B. napus) proteins were less conserved among subfamilies, but were conserved within each subfamily. A series of analyses revealed that 89.7% of the BnGRASs did not have intron insertions, and 24 specific-motifs were found at the N-terminal. A highly conserved microRNA 171 (miRNA171) target was observed specifically in the HAM subfamily across land plants. A total of 868 pairs of interaction proteins were predicted, the primary of which were transcription factors involved in transcriptional regulation and signal transduction. Integrated comparative analysis of GRAS genes across 26 species of algae, mosses, ferns, gymnosperms, and angiosperms revealed that this gene family originated in early mosses and was classified into 19 subfamilies, 14 of which may have originated prior to bryophyte evolution. RNA-Seq analysis demonstrated that most BnGRASs were widely expressed in different tissues/organs at different stages in B. napus, and 24 BnGRASs were highly/specifically expressed in roots. Results from a qRT-PCR analysis suggested that two BnGRASs belonging to SCR and LISCL subfamilies potentially have important roles in the stress response of roots.

Genome-wide analysis of GRAS transcription factor gene family in Gossypium hirsutum L.

BACKGROUND: Cotton is a major fiber and oil crop worldwide. Cotton production, however, is often threatened by abiotic environmental stresses. GRAS family proteins are among the most abundant transcription factors in plants and play important roles in regulating root and shoot development, which can improve plant resistance to abiotic stresses. However, few studies on the GRAS family have been conducted in cotton. Recently, the G. hirsutum genome sequences have been released, which provide us an opportunity to analyze the GRAS family in G. hirsutum. RESULTS: In total, 150 GRAS proteins from G. hirsutum were identified. Phylogenetic analysis showed that these GRAS protins could be classified into 14 subfamilies including SCR, DLT, OS19, LAS, SCL4/7, OS4, OS43, DELLA, PAT1, SHR, HAM, SCL3, LISCL and G_GRAS. The gene structure and motif distribution analysis of the GRAS members in G. hirsutum revealed that many genes of the SHR subfamily have more than one intron, which maybe a kind of form in the evolution of plant by obtaining or losing introns. Chromosomal location and duplication analysis revealed that segment and tandem duplication maybe the reasons of the expension of the GRAS family in cotton. Gene expression analysis confirmed the expression level of GRAS members were up-regulated under different abiotic stresses, suggesting that their possible roles in response to stresses. What's more, higher expression level in root, stem, leaf and pistil also indicated these genes may have effect on the development and breeding of cotton. CONCLUSIONS: This study firstly shows the comprehensive analysis of GRAS members in G. hirsutum. Our results provide important information about GRAS family and a framework for stress-resistant breeding in G. hirsutum.

Genome-wide identification, phylogeny and expression analyses of SCARECROW-LIKE(SCL) genes in millet (Setaria italica).

As a member of the GRAS gene family, SCARECROW-LIKE (SCL) genes encode transcriptional regulators that are involved in plant information transmission and signal transduction. In this study, 44 SCL genes including two SCARECROW genes in millet were identified to be distributed on eight chromosomes, except chromosome 6. All the millet genes contain motifs 6-8, indicating that these motifs are conserved during the evolution. SCL genes of millet were divided into eight groups based on the phylogenetic relationship and classification of Arabidopsis SCL genes. Several putative millet orthologous genes in Arabidopsis, maize and rice were identified. High throughput RNA sequencing revealed that the expressions of millet SCL genes in root, stem, leaf, spica, and along leaf gradient varied greatly. Analyses combining the gene expression patterns, gene structures, motif compositions, promoter cis-elements identification, alternative splicing of transcripts and phylogenetic relationship of SCL genes indicate that the these genes may play diverse functions. Functionally characterized SCL genes in maize, rice and Arabidopsis would provide us some clues for future characterization of their homologues in millet. To the best of our knowledge, this is the first study of millet SCL genes at the genome wide level. Our work provides a useful platform for functional analysis of SCL genes in millet, a model crop for C4 photosynthesis and bioenergy studies.

The elicitor-responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23.

We show that a rice GRAS family protein, CIGR2, is a bonafide transcriptional activator, and through this function, targets the B-type heat shock protein-encoding gene OsHsf23 (Os09g0456800). CIGR2 (Os07g0583600) is an N-acetylchitooligosaccharide elicitor-responsive gene whose activity, through the direct transcriptional control of OsHsf23, is required for mediating hypersensitive cell death activation during pathogen infection. RNAi lines of CIGR2 and OsHsf23 similarly exhibited the higher level of granulation in the epidermal cells of leaf sheath inoculated with an avirulent isolate of rice blast fungus. Interestingly, we did not observe altered levels of resistance, suggesting that CIGR2 suppresses excessive cell death in the incompatible interaction with blast fungus via activation of OsHsf23.