Huanglongbing (HLB), a bacterial disease caused by Candidatus Liberibacter asiaticus (CLas), is a major threat to the citrus industry. In a previous study conducted by our laboratory, several citrus transgenic trees expressing the Arabidopsis thaliana NPR1 (AtNPR1) gene remained HLB-free when grown in a field site under high HLB disease pressure. To determine the molecular mechanisms behind AtNPR1-mediated tolerance to HLB, a transcriptome analysis was performed using AtNPR1 overexpressing transgenic trees and non-transgenic trees as control, from which we identified 57 differentially expressed genes (DEGs). Data mining revealed the enhanced transcription of genes encoding pathogen-associated molecular patterns (PAMPs), transcription factors, leucine-rich repeat receptor kinases (LRR-RKs), and putative ankyrin repeat-containing proteins. These proteins were highly upregulated in the AtNPR1 transgenic line compared to the control plant. Furthermore, analysis of protein–protein interactions indicated that AtNPR1 interacts with CsNPR3 and CsTGA5 in the nucleus. Our results suggest that AtNPR1 positively regulates the innate defense mechanisms in citrus thereby boosting resistance and effectively protecting the plant against HLB.

Lanelate navel orange (Citrus sinensis Osbeck) is a late-ripening citrus cultivar increasingly planted in China. The physiological disorder juice sac granulation often occurs in the fruit before harvest, but the physiological and molecular mechanisms underlying this disorder remain elusive. In this study, we found that fruit granulation of the late-ripening navel orange in the Three Gorges area is mainly caused by the low winter temperature in high altitude areas. Besides, dynamic changes of water content in the fruit after freezing were clarified. The granulation of fruit juice sacs resulted in increases in cell wall cellulose and decreases in soluble solid content, and the cells gradually became shrivelled and hollow. Meanwhile, the contents of pectin, cellulose, and lignin in juice sac increased with increasing degrees of fruit granulation. The activities of pectin methylesterase (PME) and the antioxidant enzymes peroxidase (POD), superoxide dismutase, and catalase increased, while those of polygalacturonase (PG) and cellulose (CL) decreased. Furthermore, a total of 903 differentially expressed genes were identified in the granulated fruit as compared with non-disordered fruit using RNA-sequencing, most of which were enriched in nine metabolic pathways, and qRT-PCR results suggested that the juice sac granulation is closely related to cell wall metabolism. In addition, the expression of PME involved in pectin decomposition was up-regulated, while that of PG was down-regulated. Phenylalanine ammonia lyase (PAL), cinnamol dehydrogenase (CAD), and POD related to lignin synthesis were up-regulated, while CL involved in cellulose decomposition was down-regulated. The expression patterns of these genes were in line with those observed in low-temperature treatment as revealed by qRT-PCR, further confirming that low winter temperature is associated with the fruit granulation of late-ripening citrus. Accordingly, low temperature would aggravate the granulation by affecting cell wall metabolism of late-ripening citrus fruit.

Background: Cold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca²⁺-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus? Results: Eight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin ‘Guijing2501’ (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content. Conclusions: In this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars.

Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying regulatory mechanisms of molecular Cd tolerance are of substantial interest. Library screening for genes related to cadmium tolerance resulted in the discovery of a gene resembling the rubber elongation factor gene and here it is designated as SaREFl based on the preliminary bioinformatics analysis. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, Cd tolerance was enhanced in Arabidopsis plants expressing SaREFl with improvement in the fresh weight and root length of seedlings, as well as physiological indexes of adult plants. The investigation of gene expression profiles exposed to four different heavy metal treatments found that SaREFl exhibited different modes of transcriptional regulation upon heavy metal excess responding most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Screening a S. alfredii Hance cDNA library using the yeast two-hybrid technology with SaREFl as a bait led to the identification of five target genes possibly interacting with SaREFl and a gene named prenylated Rab acceptor 1 (PRA1) domain protein which was detected with a high frequency. Subcellular localization revealed that SaREFl mainly distributed in the cytosol. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants.

Chestnut (Castanea mollissima Blume) is an important economic tree owing to its tasty fruit and adaptability to environmental stresses, especially drought. Currently, there is limited information about non-specific lipid transfer protein (nsLTP) genes that respond to abiotic stress in chestnuts. Here, a chestnut nsLTP, named CmnsLTP6.9, was identified and analyzed. The results showed that the CmnsLTP6.9 protein localized in the extracellular matrix had two splicing variants (CmnsLTP6.9L and CmnsLTP6.9S). Compared with CmnsLTP6.9L, CmnsLTP6.9S had an 87 bp deletion in the 5′-terminal. Overexpression of CmnsLTP6.9L in Arabidopsis enhanced tolerance to osmotic and drought stress. Upon exposure to osmotic and drought treatment, CmnsLTP6.9L could increase reactive oxygen species (ROS)-scavenging enzyme activity, alleviating ROS damage. However, CmnsLTP6.9S-overexpressing lines showed no significant differences in phenotype, ROS content, and related enzyme activities compared with the wild type (WT) under osmotic and drought treatment. Moreover, lipid metabolism analysis confirmed that, unlike CmnsLTP6.9S, CmnsLTP6.9L mainly altered and upregulated many fatty acyls and glycerophospholipids, which implied that CmnsLTP6.9L and CmnsLTP6.9S played different roles in lipid transference in the chestnut. Taken together, we analyzed the functions of CmnsLTP6.9L and CmnsLTP6.9S, and demonstrated that CmnsLTP6.9L enhanced drought and osmotic stress tolerance through ROS scavenging and lipid metabolism.