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SUMMARY: Citrus is a model plant for studying adventitious embryos, a form of asexual reproduction controlled by a single dominant gene, RWP. This gene has been identified as the causal gene for nucellar embryogenesis, but its function has not yet been fully understood. In this study, we used the fast‐growing Fortunella hindsii as a system to explore chromatin accessibility during the nucellar embryony initiation, emphasizing elevated chromatin accessibility in polyembryonic (PO) genotypes compared to monoembryonic ones (MO). Notably, a higher level of accessible chromatin was observed in one allele of the promoter region of FhRWP, consistent with increased expression of the allele carrying the causal structural variant. By independently performing RNAi and gene editing experiments on PO genotypes, we found the downregulation of FhRWP expression could reduce the number of nucellar embryos, while its knockout resulted in abnormal axillary bud development. In overexpression experiments, FhRWP was identified as having the unique capability of inducing the embryogenic callus formation in MO stem segments, possibly through the regulation of the WUS‐CLV signaling network and the ABA and cytokinin pathway, marking the inaugural demonstration of FhRWP's potential to reignite somatic cells' embryogenic fate. This study reveals the pleiotropic function of RWP in citrus and constructs a regulatory network during adventitious embryo formation, providing a new tool for bioengineering applications in plant regeneration. Significance Statement: FhRWP plays a central role in regulating multiple developmental phenotypes, including axillary bud differentiation, nucellar embryo formation, and somatic embryogenesis in citrus. [ABSTRACT FROM AUTHOR]

Dwarfing is a pivotal agronomic trait affecting both yield and quality. Citrus species exhibit substantial variation in plant height, among which internode length is a core element. However, the molecular mechanism governing internode elongation remains unclear. Here, we unveiled that the transcriptional cascade consisting of B‐BOX DOMAIN PROTEIN 22 (BBX22) and ELONGATED HYPOCOTYL 5 (HY5) finely tunes plant height and internode elongation in citrus. Loss‐of‐function mutations of BBX22 in an early‐flowering citrus (Citrus hindsii "SJG") promoted internode elongation and reduced pigment accumulation, whereas ectopic expression of BBX22 in SJG, sweet orange (C. sinensis), pomelo (C. maxima) or heterologous expression of BBX22 in tomato (Solanum lycopersicum) significantly decreased internode length. Furthermore, exogenous application of gibberellin A3 (GA3) rescued the shortened internode and dwarf phenotype caused by BBX22 overexpression. Additional experiments revealed that BBX22 played a dual role in regulation internode elongation and pigmentation in citrus. On the one hand, it directly bound to and activated the expression of HY5, GA metabolism gene (GA2 OXIDASE 8, GA2ox8), carotenoid biosynthesis gene (PHYTOENE SYNTHASE 1, PSY1) and anthocyanin regulatory gene (Ruby1, a MYB DOMAIN PROTEIN). On the other hand, it acted as a cofactor of HY5, enhancing the ability of HY5 to regulate target genes expression. Together, our results reveal the critical role of the transcriptional cascade consisting of BBX22 and HY5 in controlling internode elongation and pigment accumulation in citrus. Unraveling the crosstalk regulatory mechanism between internode elongation and fruit pigmentation provides key genes for breeding of novel types with both dwarf and health‐beneficial fortification in citrus. [ABSTRACT FROM AUTHOR]

Summary: Self‐incompatibility (SI) is a widespread prezygotic mechanism for flowering plants to avoid inbreeding depression and promote genetic diversity. Citrus has an S‐RNase‐based SI system, which was frequently lost during evolution. We previously identified a single nucleotide mutation in Sm‐RNase, which is responsible for the loss of SI in mandarin and its hybrids. However, little is known about other mechanisms responsible for conversion of SI to self‐compatibility (SC) and we identify a completely different mechanism widely utilized by citrus. Here, we found a 786‐bp miniature inverted‐repeat transposable element (MITE) insertion in the promoter region of the FhiS2‐RNase in Fortunella hindsii Swingle (a model plant for citrus gene function), which does not contain the Sm‐RNase allele but are still SC. We demonstrate that this MITE plays a pivotal role in the loss of SI in citrus, providing evidence that this MITE insertion prevents expression of the S‐RNase; moreover, transgenic experiments show that deletion of this 786‐bp MITE insertion recovers the expression of FhiS2‐RNase and restores SI. This study identifies the first evidence for a role for MITEs at the S‐locus affecting the SI phenotype. A family‐wide survey of the S‐locus revealed that MITE insertions occur frequently adjacent to S‐RNase alleles in different citrus genera, but only certain MITEs appear to be responsible for the loss of SI. Our study provides evidence that insertion of MITEs into a promoter region can alter a breeding strategy and suggests that this phenomenon may be broadly responsible for SC in species with the S‐RNase system. [ABSTRACT FROM AUTHOR]

Summary: Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA‐mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism‐related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid‐driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5‐CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5‐CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5‐CsbZIP44 precisely modulates ABA signal‐mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops. [ABSTRACT FROM AUTHOR]

Blood orange is generally recognized to accumulate anthocyanins in its fruit pulp in a cold‐inducible manner. We observed that the fruit peel of blood orange can also accumulate anthocyanins under ample light conditions. Interestingly, purple pummelo can accumulate anthocyanins only in its fruit peel but not in its pulp. The mechanism underlying the tissue specificity of anthocyanin accumulation in citrus is unknown. Here, we show that the active promoter of Ruby1, a key activator of anthocyanin biosynthesis, is also light inducible in addition to its already known cold inducibility in blood orange. Electrophoretic mobility shift assays and transient expression assays showed that HY5 positively regulated the transcription of Ruby1 by binding to the G‐box motif (CACGTC). The tissue specificity of anthocyanin accumulation in the peel of purple pummelo may be due to the lack of a low temperature responsive element and a MYC binding site, which were shown to be involved in cold inducibility of CsRuby1 in blood orange by insertion of a long terminal repeat type retrotransposon in the promoter. These results bring new insights into the regulatory mechanism of anthocyanin biosynthesis in response to environmental stimuli and provide cis‐elements for genetic improvement of anthocyanin‐stable fruits rich in antioxidant metabolites. The accumulation of anthocyanins in fruit peel of blood orange in response to light is regulated by the G‐box (CACGTC) within the promoter of CsRuby1. On the other hand, cold‐inducible accumulation of anthocyanins in blood orange is regulated by the LTR region of the retrotransposon‐containing promoter of CsRuby1. The accumulation of anthocyanins in fruit peel of blood orange in response to light is regulated by the G‐box (CACGTC) within the promoter of CsRuby1. On the other hand, cold inducible accumulation of anthocyanins in blood orange is regulated by the LTR region of the retrotransposon‐containing promoter of CsRuby1. [ABSTRACT FROM AUTHOR]

Summary: Long intergenic non‐coding RNAs (lincRNAs) play important roles in various biological processes in plants. However, little information is known about the evolutionary characteristics of lincRNAs among closely related plant species. Here, we present a large‐scale comparative study of lincRNA transcription patterns in nine citrus species. By strand‐specific RNA‐sequencing, we identified 18 075 lincRNAs (14 575 lincRNA loci) from 34 tissue samples. The results indicated that the evolution of lincRNA transcription is more rapid than that of mRNAs. In total, 82.8–97.6% of sweet orange (Citrus sinensis) lincRNA genes were shown to have homologous sequences in other citrus genomes. However, only 15.5–28.8% of these genes had transcribed homologous lincRNAs in these citrus species, presenting a strong contrast to the high conservation of mRNA transcription (81.6–84.7%). Moreover, primitive and modern citrus lincRNAs were preferentially expressed in reproductive and vegetative organs, respectively. Evolutionarily conserved lincRNAs showed higher expression levels and lower tissue specificity than species‐specific lincRNAs. Notably, we observed a similar tissue expression pattern of homologous lincRNAs in sweet orange and pummelo (Citrus grandis), suggesting that these lincRNAs may be functionally conserved and selectively maintained. We also identified and validated a lincRNA with the highest expression in fruit that acts as an endogenous target mimic (eTM) of csi‐miR166c, and two lincRNAs that act as a precursor and target of csi‐miR166c, respectively. These lincRNAs together with csi‐miR166c could form an eTM166‐miR166c‐targeted lincRNA regulatory network that possibly affects citrus fruit development. Significance Statement: Here, 18 075 lincRNAs have been identified from 34 tissue samples from nine citrus species. The data uncover the rapid turnover of lincRNA transcription among different citrus species and highlights the potential contribution of the eTM166‐miR166‐targeted lincRNA regulatory network to fruit development. [ABSTRACT FROM AUTHOR]

- a cross between a sweet orange and I C. trifoliata i L., yet another Chinese species); and, in turn, (i) its cross with I C. japonica i , the citrangequat ( I C i . 186: 27. 2021), I Citrus reticulata i was named from plants cultivated in the Philippines, which plants, like all apomictic "mandarins" in international commerce, are part of the I C i . With the formerly common use of I Citrus nobilis i Lour. for mandarin terminated when that name was shown to be based on a hybrid, the name I Citrus reticulata i Blanco, now shown to be suffering the same fate, was first applied to mandarins following Merrill's issuing of "illustrative" modern specimens from the Philippines for Blanco's taxa (Blanco having left no known herbarium). [Extracted from the article]

Somatic embryogenesis ( SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of mi RNAs/si RNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing ( HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel mi RNAs, 130 miniature inverted-repeat transposable elements ( MITEs) derived, 94 other and 235 phased small interfering RNAs (si RNAs), as well as 203 target genes. The majority of the abundantly expressed mi RNAs/si RNAs exhibit lower expression levels in embryogenic callus ( EC) or during SE process than in nonembryogenic callus ( NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors ( TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, mi RNA- and si RNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved mi RNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE. [ABSTRACT FROM AUTHOR]