Roles of Brossinosteroids Signaling in Biotic and Abiotic Stresses.

Brassinosteroids (BRs) are a group of plant-specific steroidal phytohormones, which play fundamental roles in regulating diverse aspects of the plant life cycle including plant growth, development, and stress response. Extensive studies have demonstrated that BRs signaling is involved in plant innate immunity as well as the response to environmental stimuli including extreme temperatures, saline-alkali, and drought. In addition, that the BRs signal interacts with other immune-related signals, constructing a complex signal network to regulate plant-microbe interactions and adaptation to adverse environments, has also been preliminarily explored. A timely and updated review on these progresses is of great significance for understanding BRs functions, improving the BRs regulation network and breeding disease-resistant crops, meantime with higher tolerance to abiotic stresses. Here, we mainly focus on the latest advances in the BRs signal which regulate plant defense and tolerance to abiotic and biotic stresses and then further highlight the crosstalk between the BRs signal and other immune-related or stress response pathways, with an aim to use them to improve crops by transgenic technologies.

Comparative analysis of the potential physiological and molecular mechanisms involved in the response to root zone hypoxia in two rootstock seedlings of the Chinese bayberry via transcriptomic analysis.

The negative effects of waterlogging can be effectively improved through the use of waterlogging-resistant rootstocks. However, the underlying physiological and molecular mechanisms of Chinese bayberry (Morella rubra) rootstock tolerance to waterlogging have not yet been investigated. This study aims to unravel the molecular regulation mechanisms underlying waterlogging-tolerant rootstocks. Two rootstocks, Morella cerifera (tolerant) and Morella rubra (sensitive), were selected for root zone hypoxia treatments, assessments of hormone levels and antioxidant enzyme activity, and transcriptomic analysis. While the contents of abscisic acid (ABA) and brassinosteroid (BR) in the roots of M. rubra decreased significantly after root zone hypoxia treatment, there were no significant changes in M. cerifera. Both the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content increased in M. cerifera but were decreased in M. rubra. Transcriptome sequencing identified 1,925 (928 up- and 997 downregulated) and 733 (278 up- and 455 downregulated) differentially expressed genes (DEGs) in the two rootstocks. The gene set enrichment analysis showed that 84 gene sets were enriched after root zone hypoxia treatment, including 57 (35 up- and 22 downregulated) and 14 (five up- and nine downregulated) gene sets derived from M. cerifera and M. rubra, respectively, while the remaining 13 gene sets were shared. KEGG pathway analysis showed specific enrichment in six pathways in M. cerifera, including the mitogen-activated protein kinase (MAPK), tyrosine metabolism, glycolysis/gluconeogenesis, ribosome, cyanoamino acid metabolism, and plant-pathogen interaction pathways. Overall, these results provide preliminary insights into the molecular mechanisms of Chinese bayberry tolerance to waterlogging.

Identification of Potential Pathways of Morella cerifera Seedlings in Response to Alkali Stress via Transcriptomic Analysis.

Alkali stress, a type of abiotic stress, severely inhibits plant growth. Only a few studies have investigated the mechanism underlying the transcriptional-level response of Morella cerifera to saline-alkali stress. Based on RNA-seq technology, gene expression differences in the fibrous roots of M. cerifera seedlings exposed to low- and high-concentration alkali stress (LAS and HAS, respectively) were investigated, and the corresponding 1312 and 1532 alkali stress-responsive genes were identified, respectively. According to gene set enrichment analysis, 65 gene sets were significantly enriched. Of these, 24 gene sets were shared by both treatment groups. LAS and HAS treatment groups exhibited 9 (all downregulated) and 32 (23 downregulated) unique gene sets, respectively. The differential gene sets mainly included those involved in trehalose biosynthesis and metabolism, phospholipid translocation, and lignin catabolism. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that M. cerifera seedlings were specifically enriched in stilbenoid, diarylheptanoid, and gingerol biosynthesis; phenylalanine, tyrosine, and tryptophan biosynthesis; and sesquiterpenoid and triterpenoid biosynthesis. Moreover, the related genes involved in hormone signaling pathways and transcription factors were determined through a localization analysis of core abiotic stress pathways. These genes and their molecular mechanisms will be the focus of future research.

Identification of potential pathways associated with indole-3-butyric acid in citrus bud germination via transcriptomic analysis.

Indole-3-butyric acid (IBA) is widely used to encourage root development in cuttings of general field crops, vegetables, forest trees, fruit trees, and flowers. However, previous studies reported that IBA inhibited the germination of citrus buds via an unknown molecular mechanism. This study aimed to unravel the regulatory mechanisms underlying this inhibition. Citrus apical buds were sprayed with 100 mg ⋅ L-1 IBA. Subsequently, the plant hormone levels were analyzed, and transcriptomic analysis was performed. The results identified 3325 upregulated genes and 2926 downregulated genes in the citrus apical buds. The gene set enrichment analysis method was used to determine the Gene Ontology related to the treatment. Genes were enriched into 157 sets, including 17 upregulated sets and 140 downregulated sets, after indole butyric acid treatment. The upregulated gene sets were related to glucose import, sugar transmembrane transporter activity, and photosynthesis. The downregulated genes were mainly related to the ribosomal subunit and cell cycle process under butyric acid treatment. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed the enrichment of 11 pathways. Of note, genes related to the ribosome and proteasome pathways were significantly downregulated. Only one pathway was significantly upregulated: the autophagy pathway. Overall, these results provided insights into the molecular mechanisms underpinning the IBA-mediated inhibition of citrus bud germination inhibition. Also, the study provided a large transcriptomics dataset that could be used for further research.

Genome-wide identification and expression analysis of citrus fruitlet abscission-related polygalacturonase genes.

Polygalacturonases (PGs) encoded by a relatively large gene family are involved in plant organ abscission, but few data is available in citrus. Here, to explore the role of PGs in citrus fruitlet abscission (CFA), we have obtained 38 citrus PG (CitPG) members, based on the citrus genome sequences. The ORF length varied from 378 to 2418 bp, encoding proteins with theoretical pI and molecular mass ranging from 4.83 to 9.92 and from 13,951.71 to 85,542.28, respectively. Most CitPGs contained no less than 3 introns, suggesting a high probability of alternative splicing. Phylogenetic tree revealed that all PGs could be divided into three groups, in which 9 CitPGs, including CitPG2, CitPG3, CitPG10, CitPG24, CitPG27, CitPG29, CitPG30, CitPG33 and CitPG34 possessed a close relationship with abscission-associated PGs, indicating their role in CFA. Expression analysis further demonstrated that CitPG2, CitPG29 and CitPG34 might be involved in CFA, the expression levels of which could be induced by ethylene, inhibited by IAA and increased during CFA. The findings in this study have provided a foundation for future studies to elucidate the roles of CitPGs in CFA.

The molecular events of IAA inhibiting citrus fruitlet abscission revealed by digital gene expression profiling.

Citrus fruits possess two abscission zones (AZ), AZ A and AZ C located at the pedicel and calyx, respectively. Early citrus fruitlet abscission (CFA) exclusively occurs at AZ A. Previous data have shown that indole-3-acetic acid (IAA) could inhibit fruitlet abscission. However, its role in CFA remains vague. In this study, we first removed the ovaries of fruitlets in order to exclude their interferences. Then, the calyxes were treated with IAA, gibberellin 3 (GA3) and 6-benzylaminopurine (6-BA), respectively. The results have shown that IAA could prevent CFA from taking place, while either GA3 or 6-BA could not. When IAA concentration decreased to a value between 30 mg/L and 40 mg/L, CFA occurred, showing a concentration-dependent manner. Digital gene expression analysis revealed that 2317 corresponded to IAA treatment, of which 1226 genes were closely related to CFA. The most affected genes included those related to biosynthesis, transport and signaling of phytohormones, primarily ethylene (ET), abscisic acid (ABA) and auxin as well as protein ubiquitination, ROS response, calcium signal transduction, cell wall and transcription factors (TFs). The results obtained in this study suggested that the IAA in AZ A could suppress ethylene biosynthesis and signaling, and then inhibit abscission signaling. To our knowledge, it is the first time to reveal the key role of IAA in CFA, which will contribute to a better understanding for the mechanism underlying CFA.

Effect of salt-stress on gene expression in citrus roots revealed by RNA-seq.

Citrus, as one of the most economically important fruits worldwide, is adversely affected by salinity stress. However, its molecular mechanisms underlying salinity tolerance are still not clear. In this study, next-generation RNA-seq technology was applied to analyze the gene expression profiling of citrus roots at 3 time points over a 24-h period of salt treatment. A total of 1831 differentially expressed genes (DEGs) were identified. Among them, 1195 and 1090 DEGs were found at 4 and 24 h, of which 454 were overlapped. Based on functional annotation, the salt overly sensitive (SOS) and reactive oxygen species (ROS) signaling pathways were found to be involved. Meanwhile, we found that hormone metabolism and signaling played important roles in salt stress. In addition, a multitude of transcription factors (TFs) including WRKY, NAC, MYB, AP2/ERF, bZIP, GATA, bHLH, ZFP, SPL, CBF, and CAMTA were identified. The genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases) were also involved in salt stress. Our data not only provided a genetic resource for discovering salt tolerance-related genes, but also furthered our understanding of the molecular mechanisms underlying salt tolerance in citrus.

Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots.

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. 'MAPK cascade'), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway'), reactive oxygen species (ROS) metabolic process (e.g. 'hydrogen peroxide catabolic process') and transcription factors (e.g., 'MYB, ZFP and bZIP') were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment.

[Identification of Pummelo Cultivars Based on Hyperspectral Imaging Technology].

Existing methods for the identification of pummelo cultivars are usually time-consuming and costly, and are therefore inconvenient to be used in cases that a rapid identification is needed. This research was aimed at identifying different pummelo cultivars by hyperspectral imaging technology which can achieve a rapid and highly sensitive measurement. A total of 240 leaf samples, 60 for each of the four cultivars were investigated. Samples were divided into two groups such as calibration set (48 samples of each cultivar) and validation set (12 samples of each cultivar) by a Kennard-Stone-based algorithm. Hyperspectral images of both adaxial and abaxial surfaces of each leaf were obtained, and were segmented into a region of interest (ROI) using a simple threshold. Spectra of leaf samples were extracted from ROI. To remove the absolute noises of the spectra, only the date of spectral range 400~1000 nm was used for analysis. Multiplicative scatter correction (MSC) and standard normal variable (SNV) were utilized for data preprocessing. Principal component analysis (PCA) was used to extract the best principal components, and successive projections algorithm (SPA) was used to extract the effective wavelengths. Least squares support vector machine (LS-SVM) was used to obtain the discrimination model of the four different pummelo cultivars. To find out the optimal values of σ2 and γ which were important parameters in LS-SVM modeling, Grid-search technique and Cross-Validation were applied. The first 10 and 11 principal components were extracted by PCA for the hyperspectral data of adaxial surface and abaxial surface, respectively. There were 31 and 21 effective wavelengths selected by SPA based on the hyperspectral data of adaxial surface and abaxial surface, respectively. The best principal components and the effective wavelengths were used as inputs of LS-SVM models, and then the PCA-LS-SVM model and the SPA-LS-SVM model were built. The results showed that 99.46% and 98.44% of identification accuracy was achieved in the calibration set for the PCA-LS-SVM model and the SPA-LS-SVM model, respectively, and a 95.83% of identification accuracy was achieved in the validation set for both the PCA-LS-SVM and the SPA- LS-SVM models, which were built based on the hyperspectral data of adaxial surface. Comparatively, the results of the PCA-LS-SVM and the SPA-LS-SVM models built based on the hyperspectral data of abaxial surface both achieved identification accuracies of 100% for both calibration set and validation set. The overall results demonstrated that use of hyperspectral data of adaxial and abaxial leaf surfaces coupled with the use of PCA-LS-SVM and the SPA-LS-SVM could achieve an accurate identification of pummelo cultivars. It was feasible to use hyperspectral imaging technology to identify different pummelo cultivars, and hyperspectral imaging technology provided an alternate way of rapid identification of pummelo cultivars. Moreover, the results in this paper demonstrated that the data from the abaxial surface of leaf was more sensitive in identifying pummelo cultivars. This study provided a new method for to the fast discrimination of pummelo cultivars.

Comprehensive analysis of SAUR gene family in citrus and its transcriptional correlation with fruitlet drop from abscission zone A.

Small auxin-up RNA (SAUR) gene family is large, and the members of which can be rapidly induced by auxin and encode highly unstable mRNAs. SAUR genes are involved in various developmental and physiological processes, such as leaf senescence, fruitlet abscission, and hypocotyl development. However, their modes of action in citrus remain unknown. Hereby, a systematic analysis of SAUR gene family in citrus was conducted through a genome-wide search. In this study, a total of 70 SAUR genes, referred to as CitSAURs, have been identified in citrus. The evolutionary relationship and the intro-exon organization were analyzed, revealing strong gene conservation and the expansion of particular functional genes during plant evolution. Expression analysis showed that the major of CitSAUR genes were expressed in at least one tissue and showed distinctive expression levels, indicating the SAUR gene family play important roles in the development and growth of citrus organs. However, there were more than 20 CitSAUR genes such as CitSARU36, CitSAUR37, and CitSAUR54 exhibiting very low expression level in all tissue tested. Twenty-three out of 70 CitSAUR genes were responded to indole-3-acetic acid (IAA) treatment, of which just CitSAUR19 was down-regulated. Additionally, 14 CitSAUR genes exhibited distinct changes during fruitlet abscission, however just 5 of them including CitSAUR06, CitSAUR08, CitSAUR44, CitSAUR61, and CitSAUR64 were associated with fruitlet abscission. The current study provides basic information for the citrus SAUR gene family and will pave the way for deciphering the precise role of SAURs in citrus development and growth as well as fruitlet abscission.

The ARF, AUX/IAA and GH3 gene families in citrus: genome-wide identification and expression analysis during fruitlet drop from abscission zone A.

Completion of the whole genome sequencing of citrus enabled us to perform genome-wide identification and functional analysis of the gene families involved in agronomic traits and morphological diversity of citrus. In this study, 22 CitARF, 11 CitGH3 and 26 CitAUX/IAA genes were identified in citrus, respectively. Phylogenetic analysis revealed that all the genes of each gene family could be subdivided into three groups and showed strong evolutionary conservation. The GH3 and AUX/IAA gene families shrank and ARF gene family was highly conserved in the citrus genome after speciation from Arabidopsis thaliana. Tissue-specific expression profiles revealed that 54 genes were expressed in at least one tissue while just 5 genes including CitARF07, CitARF20, CitGH3.04, CitAUX/IAA25 and CitAUX/IAA26 with very low expression level in all tissues tested, suggesting that the CitARF, CitGH3 and CitAUX/IAA gene families played important roles in the development of citrus organs. In addition, our data found that the expression of 2 CitARF, 4 CitGH3 and 4 AUX/IAA genes was affected by IAA treatment, and 7 genes including, CitGH3.04, CitGH3.07, CitAUX/IAA03, CitAUX/IAA04, CitAUX/IAA18, CitAUX/IAA19 and CitAUX/IAA23 were related to fruitlet abscission. This study provides a foundation for future studies on elucidating the precise role of citrus ARF, GH3 and AUX/IAA genes in early steps of auxin signal transduction and open up a new opportunity to uncover the molecular mechanism underlying citrus fruitlet abscission.

Genome-wide analysis of citrus R2R3MYB genes and their spatiotemporal expression under stresses and hormone treatments.

The R2R3MYB proteins represent one of the largest families of transcription factors, which play important roles in plant growth and development. Although genome-wide analysis of this family has been conducted in many species, little is known about R2R3MYB genes in citrus, In this study, 101 R2R3MYB genes has been identified in the citrus (Citrus sinesis and Citrus clementina) genomes, which are almost equal to the number of rice. Phylogenetic analysis revealed that they could be subdivided into 21 subgroups. The evolutionary relationships and the intro-exon organizations were also analyzed, revealing strong gene conservation but also the expansions of particular functional genes during the plant evolution. Tissue-specific expression profiles showed that 95 citrus R2R3MYB genes were expressed in at least one tissue and the other 6 genes showed very low expression in all tissues tested, suggesting that citrus R2R3MYB genes play important roles in the development of all citrus organs. The transcript abundance level analysis during abiotic conditions (NaCl, abscisic acid, jasmonic acid, drought and low temperature) identified a group of R2R3MYB genes that responded to one or multiple treatments, which showed a promising for improving citrus adaptation to stresses. Our results provided an essential foundation for the future selection of the citrus R2R3MYB genes for cloning and functional dissection with an aim of uncovering their roles in citrus growth and development.

[Determination of photosynthetic pigments in citrus leaves based on hyperspectral images datas].

The effective region was segmented from the hyperspectral image of citrus leaf by threshold method with the average spectrum extracted and used to describe the corresponding leaf. Based on the different spectral pre-processing methods, the prediction models of three photosynthetic pigments (i. e., chlorophyll a, chlorophyll b, and carotenoid) were calibrated by partial least squares (PLS), BP neural network (BPNN) and least square support vector machine (LS-SVM). The LS-SVM model for chlorophyll a was established based on multiplicative scatter correction (MSC), and the correlation coefficient (Rp) and the root mean square error of prediction (RMSEP) were 0.898 3 and 0.140 4, respectively. The LS-SVM model for chlorophyll b with Rp = 0.912 3 and RMSEP = 0.042 6, was established based on standard normal variable (SNV). The PLS model for carotenoid was established with Rp = 0.712 8 and RMSEP = 0.062 4 based on moving average smoothing (MAS), but the result was no better than the other two. The results illustrated that these three photosynthetic pigments could be nondestructively and real time estimated by hyperspectral image.

Antifungal Activity of Citrus Essential Oils.

Citrus essential oils (CEOs) are a mixture of volatile compounds consisting mainly of monoterpene hydrocarbons and are widely used in the food and pharmaceutical industries because of their antifungal activities. To face the challenge of growing public awareness and concern about food and health safety, studies concerning natural biopreservatives have become the focus of multidisciplinary research efforts. In the past decades, a large amount of literature has been published on the antifungal activity of CEOs. This paper reviews the advances of research on CEOs and focuses on their in vitro and food antifungal activities, chemical compositions of CEOs, and the methods used in antifungal assessment. Furthermore, the antifungal bioactive components in CEOs and their potential mechanism of action are summarized. Finally, the applications of CEOs in the food industry are discussed in an attempt to provide new information for future utilization of CEOs in modern industries.

[Identification of different Citrus sinensis (L.) Osbeck trees varieties using Fourier transform infrared spectroscopy and hierarchical cluster analysis].

Researched on diversity of the spring leaf samples of seven different Citrus sinensis (L.) Osbeck varieties by Fourier transform infrared (FTIR) spectroscopy technology, the results showed that the Fourier transform infrared spectra of seven varieties leaves was composited by the absorption band of cellulose and polysaccharide mainly, the wave number of characteristics absorption peaks were similar at their FTIR spectra. However, there were some differences in shape of peaks and relatively absorption intensity. The conspicuous difference was presented at the region between 1 500 and 700 cm(-1) by second derivative spectra. Through the hierarchical cluster analysis (HCA) of second derivative spectra between 1 500 and 700 cm(-1), the results showed that the clustering of the different varieties of Citrus sinensis (L.) Osbeck varieties was classification according to genetic relationship. The results showed that FTIR spectroscopy combined with hierarchical cluster analysis could be used to identify and classify of citrus varieties rapidly, it was an extension method to study on early leaves of varieties orange seedlings.

[Study on synchronous correlation between fruit characteristic spectrum and the parameter of internal quality for Hamlin sweet orange fruit].

The purpose of our study is to study the relationship between the reflective spectrum of fruit and the internal quality of the orange fruit and find the suitable mark spectrum that can synchronously measure the several fruit quality index at same time to lay a foundation for the development of a rapid and nondestructive field fruit quality analysis technique by analyzing the visible-near infrared spectrum. Mature Hamlin orange (Citrus sinensis (L)cv. Hamlin sweet orange) fruits were individually mensurated for their reflective spectrum by using FieldSpec-HH spectrometer and for their contents of total soluble solid (TSS), citric acid and vitamin C (Vc) by chemistry analysis. The experiment results showed that the fruit reflectivity values (x) at 988 nm was significantly correlated to both TSS (y)(r = 0.387* *, y = 13. 957x + 5.405), TSS/acid ratio(y)(r = 0. 440* *, y = 75.120x + 37.256), and Vc(r = 0.309*). Both of TSS and Vc contends were positively correlated with the second derivatives of the reflective spectrum at 943 nm, with correlation coefficients of 0.339* and 0.355*. TSS/acid ratio was positively correlated only with the reciprocal log values of the reflective spectrum at 944 nm (r = 0.304*). The results in this study indicated that fruit quality indexes TSS, Vc and TSS/acid can be synchronously, rapidly and nondestructively field measured at the same time by the 988 or 429 nm reflective spectrum test and special regress equation operation.

[Study on the correlation of spectral characteristic and nitrogen content of the soil in citrus orchard of three Gorges Reservoir Area].

The relationship between the spectrum characteristics and nitrogen content of soils in citrus orchard of the Three Gorges Reservoir Area was studied by analyzing the visible-near infrared spectrum. The results showed that the soil reflectivity in creased lineally as the wavelength increases across the visible spectrum and reached a stable plateau in the short wavelength near-infrared region (780-1750 nm)without much fluctuation. In the long wavelength near-infrared region (1750-2400 nm) the reflec tivity of the soils was higher with higher fluctuation. There were three strong absorbance peaks around 1416, 1913 and 2209 nm, respectively, in the long wavelength infrared region. Soil available nitrogen content and total nitrogen content were positively correlated with soil light reflectivity but negatively correlated with catoptric-spectrum values reciprocal logarithm. At 541 nn of visible light region, a high positive correlation was found between the available nitrogen content and the first derivative of the soil reflective spectrum with a correlation coefficient of + 0.605** and the best fitting equation was y = 2E + 09x(2) - 3E + 06x + 890.49, where R2 = 0.5, and x is the first derivative of the soil reflective spectrum. At 1909 nm of the near-infrared long wavelength region, the correlation between the total nitrogen content and the reciprocal-log values of the reflective spectrum of the soils was the best with a correlation coefficient of -0.612**, and the best fitting equation was y = 1.3721x(2) - 2.1075x + 0.8592, where R2 = 0.4, and x is the reciprocal values of the log reflective spectrum of the soils.