WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report functional characterization of a Group III WRKY gene (FcWRKY70) from Fortunella crassifolia. FcWRKY70 was greatly induced by drought and abscisic acid, but slightly or negligibly by salt and cold. Overexpression of FcWRKY70 in tobacco (Nicotiana nudicaulis) and lemon (Citrus lemon) conferred enhanced tolerance to dehydration and drought stresses. Transgenic tobacco and lemon exhibited higher expression levels of ADC (arginine decarboxylase), and accumulated larger amount of putrescine in comparison with wild type (WT). Treatment with D-arginine, an inhibitor of ADC, caused transgenic tobacco plants more sensitive to dehydration. Knock-down of FcWRKY70 in kumquat down-regulated ADC abundance and decreased putrescine level, accompanied by compromised dehydration tolerance. The promoter region of FcADC contained two W-box elements, which were shown to be interacted with FcWRKY70. Taken together, our data demonstrated that FcWRKY70 functions in drought tolerance by, at least partly, promoting production of putrescine via regulating ADC expression.

HighlightPtrABF, a positive regulator of dehydration tolerance, is involved in stomatal development and regulates polyamine biosynthesis.

Ethylene-responsive factors (ERFs) are plant-specific transcription factors involved in cold stress response, and raffinose is known to accumulate in plants exposed to cold. However, it remains elusive whether ERFs function in cold tolerance by modulating raffinose synthesis. Here, we identified a cold-responsive PtrERF108 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-tolerant plant closely related to citrus. PtrERF108 is localized in the nucleus and has transcriptional activation activity. Overexpression of PtrERF108 conferred enhanced cold tolerance of transgenic lemon, whereas virus-induced gene silencing (VIGS)mediated knockdown of PtrERF108 in trifoliate orange greatly elevated cold sensitivity. Transcriptome profiling showed that PtrERF108 overexpression caused extensive reprogramming of genes associated with signaling transduction, physiological processes and metabolic pathways. Among them, a raffinose synthase (RafS)-encoding gene, PtrRafS, was confirmed as a direct target of PtrERF108. RafS activity and raffinose content were significantly increased in PtrERF108-overexpressing transgenic plants, but prominently decreased in the VIGS plants under cold conditions. Meanwhile, exogenous replenishment of raffinose could recover the cold tolerance of PtrERF108-silenced plants, whereas VIGS-mediated knockdown of PtrRafS resulted in cold-sensitive phenotype. Taken together, the current results demonstrate that PtrERF108 plays a positive role in cold tolerance by modulation of raffinose synthesis via regulating PtrRafS. Our findings reveal a new transcriptional module composed of ERF108-RafS underlying cold-induced raffinose accumulation in plants.

Apomixis, or asexual seed formation is prevalent in the Citrinae via a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in the Citrinae, a chromosome-level genome of Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinae accessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinae diversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella, we found that apomixis of some varieties originated via introgression. In apomictic Fortunella, the locus associated with apomixis contains the FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus or Fortunella due to either two or three Miniature inverted transposon element (MITE) insertions. A transcription factor FhARID, encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinae and has potential ramifications for citrus breeding.

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in ‘Guiyou No. 1’ pummelo (Citrus maxima), which is a spontaneous mutant of ‘Shatian’ pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of ‘Guiyou No. 1’. Segregation ratios of the S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA contribute to the loss of SI in ‘Guiyou No. 1’. Furthermore, we report a phased assembly of the ‘Guiyou No. 1’ pummelo genome and obtained two complete and well-annotated S haplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNase promoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in ‘Guiyou No. 1’ by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes.