ACC SYNTHASE4 inhibits gibberellin biosynthesis and FLOWERING LOCUS T expression during citrus flowering.

Flowering is an essential process in fruit trees. Flower number and timing have a substantial impact on the yield and maturity of fruit. Ethylene and gibberellin (GA) play vital roles in flowering, but the mechanism of coordinated regulation of flowering in woody plants by GA and ethylene is still unclear. In this study, a lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylic acid synthase gene (CiACS4) was overexpressed in Nicotiana tabacum and resulted in late flowering and increased flower number. Further transformation of citrus revealed that ethylene and starch content increased, and soluble sugar content decreased in 35S:CiACS4 lemon. Inhibition of CiACS4 in lemon resulted in effects opposite to that of 35S:CiACS4 in transgenic plants. Overexpression of the CiACS4-interacting protein ETHYLENE RESPONSE FACTOR3 (CiERF3) in N. tabacum resulted in delayed flowering and more flowers. Further experiments revealed that the CiACS4-CiERF3 complex can bind the promoters of FLOWERING LOCUS T (CiFT) and GOLDEN2-LIKE (CiFE) and suppress their expression. Moreover, overexpression of CiFE in N. tabacum led to early flowering and decreased flowers, and ethylene, starch, and soluble sugar contents were opposite to those in 35S:CiACS4 transgenic plants. Interestingly, CiFE also bound the promoter of CiFT. Additionally, GA3 and 1-aminocyclopropanecarboxylic acid (ACC) treatments delayed flowering in adult citrus, and treatment with GA and ethylene inhibitors increased flower number. ACC treatment also inhibited the expression of CiFT and CiFE. This study provides a theoretical basis for the application of ethylene to regulate flower number and mitigate the impacts of extreme weather on citrus yield due to delayed flowering.

Citrus ACC synthase CiACS4 regulates plant height by inhibiting gibberellin biosynthesis.

Dwarfism is an agronomic trait that has substantial effects on crop yield, lodging resistance, planting density, and a high harvest index. Ethylene plays an important role in plant growth and development, including the determination of plant height. However, the mechanism by which ethylene regulates plant height, especially in woody plants, remains unclear. In this study, a 1-aminocyclopropane-1-carboxylic acid synthase (ACC) gene (ACS), which is involved in ethylene biosynthesis, was isolated from lemon (Citrus limon L. Burm) and named CiACS4. Overexpression of CiACS4 resulted in a dwarf phenotype in Nicotiana tabacum and lemon and increased ethylene release and decreased gibberellin (GA) content in transgenic plants. Inhibition of CiACS4 expression in transgenic citrus significantly increased plant height compared with the controls. Yeast two-hybrid assays revealed that CiACS4 interacted with an ethylene response factor (ERF), CiERF3. Further experiments revealed that the CiACS4-CiERF3 complex can bind to the promoters of 2 citrus GA20-oxidase genes, CiGA20ox1 and CiGA20ox2, and suppress their expression. In addition, another ERF transcription factor, CiERF023, identified using yeast one-hybrid assays, promoted CiACS4 expression by binding to its promoter. Overexpression of CiERF023 in N. tabacum caused a dwarfing phenotype. CiACS4, CiERF3, and CiERF023 expression was inhibited and induced by GA3 and ACC treatments, respectively. These results suggest that the CiACS4-CiERF3 complex may be involved in the regulation of plant height by regulating CiGA20ox1 and CiGA20ox2 expression levels in citrus.

Citrus FRIGIDA cooperates with its interaction partner dehydrin to regulate drought tolerance.

Drought is a major environmental stress that severely affects plant growth and crop productivity. FRIGIDA (FRI) is a key regulator of flowering time and drought tolerance in model plants. However, little is known regarding its functions in woody plants, including citrus. Thus, we explored the functional role of the citrus FRI ortholog (CiFRI) under drought. Drought treatment induced CiFRI expression. CiFRI overexpression enhanced drought tolerance in transgenic Arabidopsis and citrus, while CiFRI suppression increased drought susceptibility in citrus. Moreover, transcriptomic profiling under drought conditions suggested that CiFRI overexpression altered the expression of numerous genes involved in the stress response, hormone biosynthesis, and signal transduction. Mechanistic studies revealed that citrus dehydrin likely protects CiFRI from stress-induced degradation, thereby enhancing plant drought tolerance. In addition, a citrus brassinazole-resistant (BZR) transcription factor family member (CiBZR1) directly binds to the CiFRI promoter to activate its expression under drought conditions. CiBZR1 also enhanced drought tolerance in transgenic Arabidopsis and citrus. These findings further our understanding of the molecular mechanisms underlying the CiFRI-mediated drought stress response in citrus.

Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells.

The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10-6 U µL-1. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.

A bZIP transcription factor (CiFD) regulates drought- and low-temperature-induced flowering by alternative splicing in citrus.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDß). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDß in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDß and CiFDα. Interestingly, CiFDα and CiFDß were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDß can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDß can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

Drought and low temperature-induced NF-YA1 activates FT expression to promote citrus flowering.

Flower induction in adult citrus is mainly regulated by drought and low temperatures. However, the mechanism of FLOWERING LOCUS T regulation of citrus flowering (CiFT) under two flower-inductive stimuli remains largely unclear. In this study, a citrus transcription factor, nuclear factor YA (CiNF-YA1), was found to specifically bind to the CiFT promoter by forming a complex with CiNF-YB2 and CiNF-YC2 to activate CiFT expression. CiNF-YA1 was induced in juvenile citrus by low temperature and drought treatments. Overexpression of CiNF-YA1 increased drought susceptibility in transgenic citrus, whereas suppression of CiNF-YA1 enhanced drought tolerance in silenced citrus plants. Furthermore, a GOLDEN2 - LIKE protein (CiFE) that interacts with CiFT protein was also isolated. Further experimental evidence showed that CiFE binds to the citrus LEAFY (CiLFY) promoter and activates its expression. In addition, the expressions of CiNF-YA1 and CiFE showed a seasonal increase during the floral induction period and were induced by artificial drought and low-temperature treatments at which floral induction occurred. These results indicate that CiNF-YA1 may activate CiFT expression in response to drought and low temperatures by binding to the CiFT promoter. CiFT then forms a complex with CiFE to activate CiLFY, thereby promoting the flowering of adult citrus.

Transcriptomic and physiological analysis reveals interplay between salicylic acid and drought stress in citrus tree floral initiation.

MAIN CONCLUSION: Salicylic acid (SA) and drought stress promote more flowering in sweet orange. The physiological response and molecular mechanism underlying stress-induced floral initiation were discovered by transcriptome profiling. Numerous flowering-regulated genes were identified, and ectopically expressed CsLIP2A promotes early flowering in Arabidopsis. Floral initiation is a critical developmental mechanism associated with external factors, and citrus flowering is mainly regulated by drought stress. However, little is known about the intricate regulatory network involved in stress-induced flowering in citrus. To understand the molecular mechanism of floral initiation in citrus, flower induction was performed on potted Citrus sinensis trees under the combined treatment of salicylic acid (SA) and drought (DR). Physiological analysis revealed that SA treatment significantly normalized the drastic effect of drought stress by increasing antioxidant enzyme activities (SOD, POD, and CAT), relative leaf water content, total chlorophyll, and proline contents and promoting more flowering than drought treatment. Analysis of transcriptome changes in leaves from different treatments showed that 1135, 2728 and 957 differentially expressed genes (DEGs) were revealed in response to DR, SD (SA + DR), and SA (SA + well water) treatments in comparison with the well watered plants, respectively. A total of 2415, 2318 and 1933 DEGs were expressed in DR, SD, and SA in comparison with water recovery, respectively. Some key flowering genes were more highly expressed in SA-treated drought plants than in DR-treated plants. GO enrichment revealed that SA treatment enhances the regulation and growth of meristem activity under drought conditions, but no such a pathway was found to be highly enriched in the control. Furthermore, we focused on various hormones, sugars, starch metabolism, and biosynthesis-related genes. The KEGG analysis demonstrated that DEGs enriched in starch sucrose metabolism and hormonal signal transduction pathways probably account for stress-induced floral initiation in citrus. In addition, a citrus LIPOYLTRANSFERSAE 2A homologous (LIP2A) gene was upregulated by SD treatment. Ectopic expression of CsLIP2A exhibited early flowering in transgenic Arabidopsis. Taken together, this study provides new insight that contributes to citrus tree floral initiation under the SA-drought scenario as well as an excellent reference for stress-induced floral initiation in woody trees.

Two citrus KNAT-like genes, CsKN1 and CsKN2, are involved in the regulation of spring shoot development in sweet orange.

Shoot-tip abortion is a very common phenomenon in some perennial woody plants and it affects the height, architecture, and branch orientation of trees; however, little is currently known about the underlying mechanisms. In this study, we identified a gene in sweet orange (Citrus sinensis) encoding a KNAT-like protein (CsKN1) and found high expression in the shoot apical meristem (SAM). Overexpression of CsKN1 in transgenic plants prolonged the vegetative growth of SAMs, whilst silencing resulted in either the loss or inhibition of SAMs. Yeast two-hybrid analysis revealed that CsKN1 interacted with another citrus KNAT-like protein (CsKN2), and overexpression of CsKN2 in lemon and tobacco caused an extreme multiple-meristem phenotype. Overexpression of CsKN1 and CsKN2 in transgenic plants resulted in the differential expression of numerous genes related to hormone biosynthesis and signaling. Yeast one-hybrid analysis revealed that the CsKN1-CsKN2 complex can bind to the promoter of citrus floral meristem gene LEAFY (CsLFY) and inhibit its expression. These results indicated that CsKN1 might prolong the vegetative growth period of SAMs by delaying flowering. In addition, an ethylene-responsive factor (CsERF) was found to bind to the CsKN1 promoter and suppresses its transcription. Overexpression of CsERF in Arabidopsis increased the contents of ethylene and reactive oxygen species, which might induce the occurrence of shoot-tip abscission. On the basis of our results, we conclude that CsKN1 and CsKN2 might work cooperatively to regulate the shoot-tip abscission process in spring shoots of sweet orange.

Genome-Wide Identification and Expression Profiling of the WOX Gene Family in Citrus sinensis and Functional Analysis of a CsWUS Member.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) are well known for their role in plant development but are rarely studied in citrus. In this study, we identified 11 putative genes from the sweet orange genome and divided the citrus WOX genes into three clades (modern/WUSCHEL(WUS), intermediate, and ancient). Subsequently, we performed syntenic relationship, intron-exon organization, motif composition, and cis-element analysis. Co-expression analysis based on RNA-seq and tissue-specific expression patterns revealed that CsWOX gene expression has multiple intrinsic functions. CsWUS homolog of AtWUS functions as a transcriptional activator and binds to specific DNA. Overexpression of CsWUS in tobacco revealed dramatic phenotypic changes, including malformed leaves and reduced gynoecia with no seed development. Silencing of CsWUS in lemon using the virus-induced gene silencing (VIGS) system implied the involvement of CsWUS in cells of the plant stem. In addition, CsWUS was found to interact with CsCYCD3, an ortholog in Arabidopsis (AtCYCD3,1). Yeast one-hybrid screening and dual luciferase activity revealed that two TFs (CsRAP2.12 and CsHB22) bind to the promoter of CsWUS and regulate its expression. Altogether, these results extend our knowledge of the WOX gene family along with CsWUS function and provide valuable findings for future study on development regulation and comprehensive data of WOX members in citrus.

High-Density Genetic Map Construction and Identification of QTLs Controlling Leaf Abscission Trait in Poncirus trifoliata.

A high-density genetic linkage map is essential for genetic and genomic studies including QTL mapping, genome assembly, and comparative genomic analysis. Here, we constructed a citrus high-density linkage map using SSR and SNP markers, which are evenly distributed across the citrus genome. The integrated linkage map contains 4163 markers with an average distance of 1.12 cM. The female and male linkage maps contain 1478 and 2976 markers with genetic lengths of 1093.90 cM and 1227.03 cM, respectively. Meanwhile, a genetic map comparison demonstrates that the linear order of common markers is highly conserved between the clementine mandarin and Poncirus trifoliata. Based on this high-density integrated citrus genetic map and two years of deciduous phenotypic data, two loci conferring leaf abscission phenotypic variation were detected on scaffold 1 (including 36 genes) and scaffold 8 (including 107 genes) using association analysis. Moreover, the expression patterns of 30 candidate genes were investigated under cold stress conditions because cold temperature is closely linked with the deciduous trait. The developed high-density genetic map will facilitate QTL mapping and genomic studies, and the localization of the leaf abscission deciduous trait will be valuable for understanding the mechanism of this deciduous trait and citrus breeding.

A MADS-Box Gene CiMADS43 Is Involved in Citrus Flowering and Leaf Development through Interaction with CiAGL9.

MADS-box genes are involved in various developmental processes including vegetative development, flower architecture, flowering, pollen formation, seed and fruit development. However, the function of most MADS-box genes and their regulation mechanism are still unclear in woody plants compared with model plants. In this study, a MADS-box gene (CiMADS43) was identified in citrus. Phylogenetic and sequence analysis showed that CiMADS43 is a GOA-like Bsister MADS-box gene. It was localized in the nucleus and as a transcriptional activator. Overexpression of CiMADS43 promoted early flowering and leaves curling in transgenic Arabidopsis. Besides, overexpression or knockout of CiMADS43 also showed leaf curl phenotype in citrus similar to that of CiMADS43 overexpressed in Arabidopsis. Protein-protein interaction found that a SEPALLATA (SEP)-like protein (CiAGL9) interacted with CiMADS43 protein. Interestingly, CiAGL9 also can bind to the CiMADS43 promoter and promote its transcription. Expression analysis also showed that these two genes were closely related to seasonal flowering and the development of the leaf in citrus. Our findings revealed the multifunctional roles of CiMADS43 in the vegetative and reproductive development of citrus. These results will facilitate our understanding of the evolution and molecular mechanisms of MADS-box genes in citrus.

Comparative analysis of the transcriptome, methylome, and metabolome during pollen abortion of a seedless citrus mutant.

KEY MESSAGE: Pollen abortion could be mainly attributed to abnormal meiosis in the mutant. Multiomics analysis uncovered significant epigenetic variations between the mutant and its wild type during the pollen abortion process. Male sterility caused by aborted pollen can result in seedless fruit. A seedless Ponkan mandarin mutant (bud sport) was used to compare the transcriptome, methylome, and metabolome with its progenitor to understand the mechanism of citrus pollen abortion. Cytological observations showed that the anther of the mutant could form microspore mother cells, although the microspores failed to develop fertile pollen at the anther dehiscence stage. Based on pollen phenotypic analysis, pollen abortion could be mainly attributed to abnormal meiosis in the mutant. A transcriptome analysis uncovered the molecular mechanisms underlying pollen abortion between the mutant and its wild type. A total of 5421 differentially expressed genes were identified, and some of these genes were involved in the meiosis, hormone biosynthesis and signaling, carbohydrate, and flavonoid pathways. A total of 50,845 differentially methylated regions corresponding to 15,426 differentially methylated genes in the genic region were found between the mutant and its wild type by the methylome analysis. The expression level of these genes was negatively correlated with their methylation level, especially in the promoter regions. In addition, 197 differential metabolites were identified between the mutant and its wild type based on the metabolome analysis. The transcription and metabolome analysis further indicated that the expression of genes in the flavonoid, carbohydrate, and hormone metabolic pathways was significantly modulated in the pollen of the mutant. These results indicated that demethylation may alleviate the silencing of carbohydrate genes in the mutant, resulting in excessive starch and sugar hydrolysis and thereby causing pollen abortion in the mutant.

The Protective Roles of Folic Acid in Preventing Diabetic Retinopathy Are Potentially Associated with Suppressions on Angiogenesis, Inflammation, and Oxidative Stress.

This study aimed to evaluate the therapeutic effect of folic acid (FA) on diabetic retinopathy (DR) in a genetic mouse model of obese type 2 diabetes mellitus (T2D). C57BL/KsJ-db/db (db/db) T2D mice were divided into control, FA, metformin (MET), and FA plus MET groups (n = 10/group). Serum levels of glucose, glycated hemoglobin, and insulin were determined weekly. The retinal thickness was measured using optical coherence tomography (OCT) at 4 weeks after treatments. The retinal expression and serum levels of vascular formation, inflammation, and oxidative stress-associated molecules were examined. Our results demonstrated that FA, but not MET, played a protective role against retinal thinning in the early stage of DR in db/db mice, although FA did not exhibit antihyperglycemic effect. In addition, retinal expression and serum levels of a panel of molecules associated with angiogenesis (CD31 and VEGFR), inflammation (IL-1ß and NLRP3), and oxidative stress (3-NT, 4-HNE, Vav2, and NOX4) were significantly downregulated in FA-treated diabetic mice compared with those in saline-treated controls. Furthermore, the serum level of homocysteine was also markedly decreased following FA treatments. These findings suggest that through potential suppressions on angiogenesis, inflammation, and oxidative stress, FA may serve as a potential therapeutic agent against DR.

Association analysis of polymorphisms of G protein-coupled receptor 54 gene exons with reproductive traits in Jiaxing Black sows.

OBJECTIVE: The aim of this study was to detect single nucleotide polymorphisms (SNP) of G protein-coupled receptor 54 (GPR54) gene and explore association of this candidate gene with reproductive traits in Jiaxing Black sows. METHODS: Six pairs of primers of the gene were designed to amplify all exons thus sequences of which were detected by means of direct sequencing and then SNP loci were scanned. The effects of SNPs on total number of piglets born (TNB), number of piglets born alive (NBA), number of still born piglets (NSB), and litter weight at birth (LWB) of Jiaxing Black sows were analyzed. RESULTS: Three SNP loci, including T3739C, C3878T and T6789C, were identified via comparison of sequencing and two genotypes (AB, BB) at each SNP site were observed. T3739C resulted in the change of amino acid (Leu→Pro) in corresponding protein, and C3878T resulted in synonymous mutation (Ile→Ile). Statistical results demonstrated that allele B was the preponderant allele at the three SNP loci and Genotype BB was the preponderant genotype. Meanwhile, Chi-Square test of these three SNPs indicated that all mutation sites fitted in Hardy-Weinberg equilibrium (p>0.05). For GPR54-T3739C locus, Jiaxing Black sows with genotype BB had 1.23 TNB and 1.28 NBA (p<0.01) that were more than those with genotype AB, respectively. Jiaxing Black sows that had the first two parities with genotype BB had additional 2.23 TNB, 2.27 NBA (p<0.01), and 1.94 LWB (p<0.05) compared to those with genotype AB, respectively. However, for other two loci, no significant difference was found between TNB, NBA, NSB, and LWB, and different genotypes of Jiaxing Black sows. CONCLUSION: In conclusion, the polymorphisms of GPR54-T3739C locus were significantly associated to TNB, NBA, and LWB and could be used as a potential genetic marker to improve reproductive function of Jiaxing black sows.

Genome-wide genetic variation and comparison of fruit-associated traits between kumquat (Citrus japonica) and Clementine mandarin (Citrus clementina).

KEY MESSAGE: The comprehensive genetic variation of two citrus species were analyzed at genome and transcriptome level. A total of 1090 differentially expressed genes were found during fruit development by RNA-sequencing. Fruit size (fruit equatorial diameter) and weight (fresh weight) are the two most important components determining yield and consumer acceptability for many horticultural crops. However, little is known about the genetic control of these traits. Here, we performed whole-genome resequencing to reveal the comprehensive genetic variation of the fruit development between kumquat (Citrus japonica) and Clementine mandarin (Citrus clementina). In total, 5,865,235 single-nucleotide polymorphisms (SNPs) and 414,447 insertions/deletions (InDels) were identified in the two citrus species. Based on integrative analysis of genome and transcriptome of fruit, 640,801 SNPs and 20,733 InDels were identified. The features, genomic distribution, functional effect, and other characteristics of these genetic variations were explored. RNA-sequencing identified 1090 differentially expressed genes (DEGs) during fruit development of kumquat and Clementine mandarin. Gene Ontology revealed that these genes were involved in various molecular functional and biological processes. In addition, the genetic variation of 939 DEGs and 74 multiple fruit development pathway genes from previous reports were also identified. A global survey identified 24,237 specific alternative splicing events in the two citrus species and showed that intron retention is the most prevalent pattern of alternative splicing. These genome variation data provide a foundation for further exploration of citrus diversity and gene-phenotype relationships and for future research on molecular breeding to improve kumquat, Clementine mandarin and related species.

Transcriptome-wide identification and functional prediction of novel and flowering-related circular RNAs from trifoliate orange (Poncirus trifoliata L. Raf.).

MAIN CONCLUSION: A total of 558 potential circular RNAs (circRNAs) were identified in citrus, and these were analyzed and compared. One hundred seventy-six differentially expressed circRNAs were identified in two genotypes of trifoliate orange. Circular RNAs (circRNAs) play diverse roles in transcriptional control and microRNA (miRNA) function. However, little information is known about circRNAs in citrus. To identify citrus circRNAs and investigate their functional roles, high-throughput sequencing of precocious trifoliate orange (an early-flowering trifoliate orange mutant, Poncirus trifoliata L. Raf.) and its wild type was performed. A total of 558 potential circRNAs were identified by bioinformatic analysis, and 86.02% of these were sense-overlapping circRNAs. Their sequence features, alternative circularization, and other characteristics were investigated in this study. Compared with the wild type, 176 circRNAs were identified as differentially expressed circRNAs, 61 were significantly up-regulated and 115 were down-regulated in precocious trifoliate orange, indicating that they may play an important role in the early flowering process. Alternative circularization and differential expression of some circRNAs were verified by Sanger sequencing and real-time polymerase chain reaction. The functions of differentially expressed circRNAs and their host genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. We found that many differentially expressed circRNAs had abundant miRNA binding sites: 29 circRNAs were found to act as the 16 miRNA targets. Overall, these results will help to reveal the biological functions of circRNAs in growth and development of citrus.

Aux/IAA Gene Family in Plants: Molecular Structure, Regulation, and Function.

Auxin plays a crucial role in the diverse cellular and developmental responses of plants across their lifespan. Plants can quickly sense and respond to changes in auxin levels, and these responses involve several major classes of auxin-responsive genes, including the Auxin/Indole-3-Acetic Acid (Aux/IAA) family, the auxin response factor (ARF) family, small auxin upregulated RNA (SAUR), and the auxin-responsive Gretchen Hagen3 (GH3) family. Aux/IAA proteins are short-lived nuclear proteins comprising several highly conserved domains that are encoded by the auxin early response gene family. These proteins have specific domains that interact with ARFs and inhibit the transcription of genes activated by ARFs. Molecular studies have revealed that Aux/IAA family members can form diverse dimers with ARFs to regulate genes in various ways. Functional analyses of Aux/IAA family members have indicated that they have various roles in plant development, such as root development, shoot growth, and fruit ripening. In this review, recently discovered details regarding the molecular characteristics, regulation, and protein-protein interactions of the Aux/IAA proteins are discussed. These details provide new insights into the molecular basis of the Aux/IAA protein functions in plant developmental processes.

Involvements of PCD and changes in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis).

BACKGROUND: Citrus shoot tips abscise at an anatomically distinct abscission zone (AZ) that separates the top part of the shoots into basal and apical portions (citrus self-pruning). Cell separation occurs only at the AZ, which suggests its cells have distinctive molecular regulation. Although several studies have looked into the morphological aspects of self-pruning process, the underlying molecular mechanisms remain unknown. RESULTS: In this study, the hallmarks of programmed cell death (PCD) were identified by TUNEL experiments, transmission electron microscopy (TEM) and histochemical staining for reactive oxygen species (ROS) during self-pruning of the spring shoots in sweet orange. Our results indicated that PCD occurred systematically and progressively and may play an important role in the control of self-pruning of citrus. Microarray analysis was used to examine transcriptome changes at three stages of self-pruning, and 1,378 differentially expressed genes were identified. Some genes were related to PCD, while others were associated with cell wall biosynthesis or metabolism. These results strongly suggest that abscission layers activate both catabolic and anabolic wall modification pathways during the self-pruning process. In addition, a strong correlation was observed between self-pruning and the expression of hormone-related genes. Self-pruning plays an important role in citrus floral bud initiation. Therefore, several key flowering homologs of Arabidopsis and tomato shoot apical meristem (SAM) activity genes were investigated in sweet orange by real-time PCR and in situ hybridization, and the results indicated that these genes were preferentially expressed in SAM as well as axillary meristem. CONCLUSION: Based on these findings, a model for sweet orange spring shoot self-pruning is proposed, which will enable us to better understand the mechanism of self-pruning and abscission.

Transcription-related metabolic regulation in grafted lemon seedlings under magnesium deficiency stress.

Magnesium is one of the essential nutrients for plant growth, and plays a pivotal role in plant development and metabolism. Soil magnesium deficiency is evident in citrus production, which ultimately leads to failure of normal plant growth and development, as well as decreased productivity. Citrus is mainly propagated by grafting, so it is necessary to fully understand the different regulatory mechanisms of rootstock and scion response to magnesium deficiency. Here, we characterized the differences in morphological alterations, physiological metabolism and differential gene expression between trifoliate orange rootstocks and lemon scions under normal and magnesium-deficient conditions, revealing the different responses of rootstocks and scions to magnesium deficiency. The transcriptomic data showed that differentially expressed genes were enriched in 14 and 4 metabolic pathways in leaves and roots, respectively, after magnesium deficiency treatment. And the magnesium transport-related genes MHX and MRS2 may respond to magnesium deficiency stress. In addition, magnesium deficiency may affect plant growth by affecting POD, SOD, and CAT enzyme activity, as well as altering the levels of hormones such as IAA, ABA, GA3, JA, and SA, and the expression of related responsive genes. In conclusion, our research suggests that the leaves of lemon grafted onto trifoliate orange were more significantly affected than the roots under magnesium-deficient conditions, further indicating that the metabolic imbalance of scion lemon leaves was more severe.