Genome-Wide Identification and Characterization of PIN-FORMED (PIN) Gene Family Reveals Role in Developmental and Various Stress Conditions in Triticum aestivum L.

PIN-FORMED (PIN) genes play a crucial role in regulating polar auxin distribution in diverse developmental processes, including tropic responses, embryogenesis, tissue differentiation, and organogenesis. However, the role of PIN-mediated auxin transport in various plant species is poorly understood. Currently, no information is available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the PIN gene family in wheat to understand the evolution of PIN-mediated auxin transport and its role in various developmental processes and under different biotic and abiotic stress conditions. In this study, we performed genome-wide analysis of the PIN gene family in common wheat and identified 44 TaPIN genes through a homology search, further characterizing them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses led to the classification of TaPIN genes into seven different groups, providing evidence of an evolutionary relationship with Arabidopsis thaliana and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, transmembrane domains, and three-dimensional (3D) structure were also examined using various computational approaches. Cis-elements analysis of TaPIN genes showed that TaPIN promoters consist of phytohormone, plant growth and development, and stress-related cis-elements. In addition, expression profile analysis also revealed that the expression patterns of the TaPIN genes were different in different tissues and developmental stages. Several members of the TaPIN family were induced during biotic and abiotic stress. Moreover, the expression patterns of TaPIN genes were verified by qRT-PCR. The qRT-PCR results also show a similar expression with slight variation. Therefore, the outcome of this study provides basic genomic information on the expression of the TaPIN gene family and will pave the way for dissecting the precise role of TaPINs in plant developmental processes and different stress conditions.     

Identification of a Major QTL and Validation of Related Genes for Tiller Angle in Rice Based on QTL Analysis

An ideal plant architecture is an important condition to achieve high crop yields. The tiller angle is an important and complex polygenic trait of rice (Oryza sativa L.) plant architecture. Therefore, the discovery and identification of tiller angle-related genes can aid in the improvement of crop architecture and yield. In the present study, 222 SSR markers were used to establish a high-density genetic map of rice doubled haploid population, and a total of 8 quantitative trait loci (QTLs) were detected based on the phenotypic data of the tiller angle and tiller crown width over 2 years. Among them, four QTLs (qTA9, qCW9, qTA9-1, and qCW9-1) were overlapped at marker interval RM6235–RM24288 on chromosome 9 with a large effect value regarded as a stable major QTL. The selected promising related genes were further identified by relative gene expression analysis, which gives us a basis for the future cloning of these genes. Finally, OsSAURq9, which belongs to the SMALL AUXIN UP RNA (SAUR), an auxin-responsive protein family, was selected as a target gene. Overall, this work will help broaden our knowledge of the genetic control of tiller angle and tiller crown width, and this study provides both a good theoretical basis and a new genetic resource for the breeding of ideal-type rice.     

Over-Expression of Rose RrLAZY1 Negatively Regulates the Branch Angle of Transgenic Arabidopsis Inflorescence

Branch angle is a key shoot architecture trait that strongly influences the ornamental and economic value of garden plants. However, the mechanism underlying the control of branch angle, an important aspect of tree architecture, is far from clear in roses. In the present study, we isolated the RrLAZY1 gene from the stems of Rosa rugosa ‘Zilong wochi’. Sequence analysis showed that the encoded RrLAZY1 protein contained a conserved GΦL (A/T) IGT domain, which belongs to the IGT family. Quantitative real-time PCR (qRT-PCR) analyses revealed that RrLAZY1 was expressed in all tissues and that expression was highest in the stem. The RrLAZY1 protein was localized in the plasma membrane. Based on a yeast two-hybrid assay and bimolecular fluorescence complementation experiments, the RrLAZY1 protein was found to interact with auxin-related proteins RrIAA16. The over-expression of the RrLAZY1 gene displayed a smaller branch angle in transgenic Arabidopsis inflorescence and resulted in changes in the expression level of genes related to auxin polar transport and signal transduction pathways. This study represents the first systematic analysis of the LAZY1 gene family in R. rugosa. The results of this study will provide a theoretical basis for the improvement of rose plant types and molecular breeding and provide valuable information for studying the regulation mechanism of branch angle in other woody plants.     

Genome-Wide Identification,Characterization and Expression Analysis of the Chalcone Synthase Family in Maize

Members of the chalcone synthase (CHS) family participate in the synthesis of a series of secondary metabolites in plants, fungi and bacteria. The metabolites play important roles in protecting land plants against various environmental stresses during the evolutionary process. Our research was conducted on comprehensive investigation of CHS genes in maize (Zea mays L.), including their phylogenetic relationships, gene structures, chromosomal locations and expression analysis. Fourteen CHS genes (ZmCHS01–14) were identified in the genome of maize, representing one of the largest numbers of CHS family members identified in one organism to date. The gene family was classified into four major classes (classes I–IV) based on their phylogenetic relationships. Most of them contained two exons and one intron. The 14 genes were unevenly located on six chromosomes. Two segmental duplication events were identified, which might contribute to the expansion of the maize CHS gene family to some extent. In addition, quantitative real-time PCR and microarray data analyses suggested that ZmCHS genes exhibited various expression patterns, indicating functional diversification of the ZmCHS genes. Our results will contribute to future studies of the complexity of the CHS gene family in maize and provide valuable information for the systematic analysis of the functions of the CHS gene family.     

Gibberellin Oxidase Gene Family in L. chinense: Genome-Wide Identification and Gene Expression Analysis

GAox is a key enzyme for the transformation of gibberellins, and belongs to the 2-ketoglutarate dependent dioxygenase gene family (2ODD). However, a systematic analysis of GAox in the angiosperm L. chinense has not yet been reported. Here, we identified all LcGAox gene family members in L. chinense, which were classified into the three subgroups of GA20ox, C19GA2ox, and C20GA2ox. Comparison of the gene structure, conserve motifs, phylogenetic relationships, and syntenic relationships of gibberellin oxidase gene families in different species indicated that the gene functional differences may be due to the partial deletion of their domains during evolution. Furthermore, evidence for purifying selection was detected between orthologous GAox genes in rice, grape, Arabidopsis, and L. chinense. Analysis of the codon usage patterns showed that mutation pressure and natural selection might have induced codon usage bias in angiosperms; however, the LcGAox genes in mosses, lycophytes, and ambarella plants exhibited no obvious codon usage preference. These results suggested that the gibberellin oxidase genes were more primitive. The gene expression pattern was analyzed in different organs subjected to multiple abiotic stresses, including GA, abscisic acid (ABA), and chlormequat (CCC) treatment, by RNA-seq and qRT-PCR, and the stress- and phytohormone-responsive cis-elements were counted. The results showed that the synthesis and decomposition of GA were regulated by different LcGAox genes in the vegetative and reproductive organs of L. chinense, and only LcGA2ox1,4, and 7 responded to the NaCl, polyethylene glycol, 4 °C, GA, ABA, and CCC treatment in the roots, stems, and leaves of seedlings at different time periods, revealing the potential role of LcGAox in stress resistance.     

Genome-Wide Identification and Characterization of the Brassinazole-resistant (BZR) Gene Family and Its Expression in the Various Developmental Stage and Stress Conditions in Wheat (Triticum aestivum L.)

Brassinosteroids (BRs) play crucial roles in various biological processes, including plant developmental processes and response to diverse biotic and abiotic stresses. However, no information is currently available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the BZR gene family in wheat to understand the evolution and their role in diverse developmental processes and under different stress conditions. In this study, we performed the genome-wide analysis of the BZR gene family in the bread wheat and identified 20 TaBZR genes through a homology search and further characterized them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses lead to the classification of TaBZR genes into five different groups or subfamilies, providing evidence of evolutionary relationship with Arabidopsis thaliana, Zea mays, Glycine max, and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, and cis-acting regulatory elements were also examined using various computational approaches. In addition, an analysis of public RNA-seq data also shows that TaBZR genes may be involved in diverse developmental processes and stress tolerance mechanisms. Moreover, qRT-PCR results also showed similar expression with slight variation. Collectively, these results suggest that TaBZR genes might play an important role in plant developmental processes and various stress conditions. Therefore, this work provides valuable information for further elucidate the precise role of BZR family members in wheat.     

Identification of a Plastid-Localized Bifunctional Nerolidol/Linalool Synthase in Relation to Linalool Biosynthesis in Young Grape Berries

Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS) synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and nerolidol in vitro, respectively, and thus it was named VvRILinNer. However, localization experiment showed that this enzyme was only localized to chloroplasts, which indicates that VvRILinNer functions in the linalool production in vivo. The patterns of gene expression and linalool accumulation were analyzed in the berries of three grape cultivars (“Riesling”, “Cabernet Sauvignon”, “Gewurztraminer”) with significantly different levels of monoterpenoids. The VvRILinNer was considered to be mainly responsible for the synthesis of linalool at the early developmental stage. This finding has provided us with new knowledge to uncover the complex monoterpene biosynthesis in grapes.     

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.     

Transcriptomic Analysis Suggests Auxin Regulation in Dorsal-Ventral Petal Asymmetry of Wild Progenitor Sinningia speciosa

The establishment of dorsal–ventral (DV) petal asymmetry is accompanied by differential growth of DV petal size, shape, and color differences, which enhance ornamental values. Genes involved in flower symmetry in Sinningia speciosa have been identified as CYCLOIDEA (SsCYC), but which gene regulatory network (GRN) is associated with SsCYC to establish DV petal asymmetry is still unknown. To uncover the GRN of DV petal asymmetry, we identified 630 DV differentially expressed genes (DV-DEGs) from the RNA-Seq of dorsal and ventral petals in the wild progenitor, S. speciosa ‘ES’. Validated by qRT-PCR, genes in the auxin signaling transduction pathway, SsCYC, and a major regulator of anthocyanin biosynthesis were upregulated in dorsal petals. These genes correlated with a higher endogenous auxin level in dorsal petals, with longer tube length growth through cell expansion and a purple dorsal color. Over-expression of SsCYC in Nicotiana reduced petal size by regulating cell growth, suggesting that SsCYC also controls cell expansion. This suggests that auxin and SsCYC both regulate DV petal asymmetry. Transiently over-expressed SsCYC, however, could not activate most major auxin signaling genes, suggesting that SsCYC may not trigger auxin regulation. Whether auxin can activate SsCYC or whether they act independently to regulate DV petal asymmetry remains to be explored in the future.     

Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus

Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression.     

Genome-Wide Comparative Analysis of the R2R3-MYB Gene Family in Five Solanaceae Species and Identification of Members Regulating Carotenoid Biosynthesis in Wolfberry

The R2R3-MYB is a large gene family involved in various plant functions, including carotenoid biosynthesis. However, this gene family lacks a comprehensive analysis in wolfberry (Lycium barbarum L.) and other Solanaceae species. The recent sequencing of the wolfberry genome provides an opportunity for investigating the organization and evolutionary characteristics of R2R3-MYB genes in wolfberry and other Solanaceae species. A total of 610 R2R3-MYB genes were identified in five Solanaceae species, including 137 in wolfberry. The LbaR2R3-MYB genes were grouped into 31 subgroups based on phylogenetic analysis, conserved gene structures, and motif composition. Five groups only of Solanaceae R2R3-MYB genes were functionally divergent during evolution. Dispersed and whole duplication events are critical for expanding the R2R3-MYB gene family. There were 287 orthologous gene pairs between wolfberry and the other four selected Solanaceae species. RNA-seq analysis identified the expression level of LbaR2R3-MYB differential gene expression (DEGs) and carotenoid biosynthesis genes (CBGs) in fruit development stages. The highly expressed LbaR2R3-MYB genes are co-expressed with CBGs during fruit development. A quantitative Real-Time (qRT)-PCR verified seven selected candidate genes. Thus, Lba11g0183 and Lba02g01219 are candidate genes regulating carotenoid biosynthesis in wolfberry. This study elucidates the evolution and function of R2R3-MYB genes in wolfberry and the four Solanaceae species.