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.

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.

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.

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.

PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants.

A MADS-box gene was isolated using the suppressive subtractive hybridization library between early-flowering mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.). This gene is highly homologous with Arabidopsis SHORT VEGETATIVE PHASE (SVP). Based on real-time PCR and in situ hybridization during bud differentiation, PtSVP was expressed intensively in dormant tissue and vegetative meristems. PtSVP transcripts were detected in apical meristems before floral transition, then down-regulated during the transition. PtSVP expression was higher in differentiated (flower primordium) than in undifferentiated cells (apical meristems). The PtSVP expression pattern during apical meristem determination suggested that its function is not to depress flower initiation but to maintain meristem development. Transcription of PtSVP in Arabidopsis svp-41 showed partially rescued SVP function. Ectopic overexpression of PtSVP in wild-type Arabidopsis induced late flowering similar to the phenotypes induced by other SVP/StMADS-11-like genes, but transformants produced additional trichomes and floral defects, such as flower-like structures instead of carpels. Ectopic expression of PtSVP in tobacco also caused additional florets. Overexpression of PtSVP in tobacco inhibited early transition of the coflorescence and prolonged coflorescence development, thus causing additional florets at the later stage. A yeast two-hybrid assay indicated that PtSVP significantly interacted with PtAP1, a homolog of Arabidopsis APETALA1 (AP1). These findings suggest that citrus SVP homolog genes are involved in flowering time regulation and may influence inflorescence meristem identity in some conditions or genetic backgrounds. SVP homologs might have evolved among plant species, but the protein functions are conserved between Arabidopsis and citrus.

Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance.

Polyploidy is reported to show increased tolerance to environmental stress. In this work, tetraploid plants of Dioscorea zingiberensis were obtained by colchicine treatment of shoots propagated in vitro. The highest tetraploid induction rate was achieved by treatment with 0.15% colchicine for 24h. Diploid and tetraploid plants were exposed to normal (28 degrees C) and high temperature (42 degrees C) for 5d during which physiological indices were measured. Compared with diploid plants, relative electrolyte leakage and contents of malondialdehyde, superoxide anions and hydrogen peroxide were lower in tetraploids, while activities of antioxidant enzymes, such as superoxide dismutase, peroxidase, catalase, ascorbate peroxidase and glutathione reductase, were stimulated and antioxidants (ascorbic acid and glutathione) were maintained at high concentrations. These results indicate that tetraploid plants possess a stronger antioxidant defense system and increased heat tolerance.