REDUCED CHLOROPLAST COVERAGE proteins are required for plastid proliferation and carotenoid accumulation in tomato.

Increasing the amount of cellular space allocated to plastids will lead to increases in the quality and yield of crop plants. However, mechanisms that allocate cellular space to plastids remain poorly understood. To test whether the tomato (Solanum lycopersicum L.) REDUCED CHLOROPLAST COVERAGE (SlREC) gene products serve as central components of the mechanism that allocates cellular space to plastids and contribute to the quality of tomato fruit, we knocked out the four-member SlREC gene family. We found that slrec mutants accumulated lower levels of chlorophyll in leaves and fruit, accumulated lower levels of carotenoids in flowers and fruits, allocated less cellular space to plastids in leaf mesophyll and fruit pericarp cells, and developed abnormal plastids in flowers and fruits. Fruit produced by slrec mutants initiated ripening later than wild type and produced abnormal levels of ethylene and ABA. Metabolome and transcriptome analyses of slrec mutant fruit indicated that the SlREC gene products markedly influence plastid-related gene expression, primary and specialized metabolism, and the response to biotic stress. Our findings and previous work with distinct species indicate that REC proteins help allocate cellular space to plastids in diverse species and cell types and, thus, play a central role in allocating cellular space to plastids. Moreover, the SlREC proteins are required for the high-level accumulation of chlorophyll and carotenoids in diverse organs, including fruit, promote the development of plastids, and influence fruit ripening by acting both upstream and downstream of ABA biosynthesis in a complex network.

Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

Phased genomics reveals hidden somatic mutations and provides insight into fruit development in sweet orange.

Although revisiting the discoveries and implications of genetic variations using phased genomics is critical, such efforts are still lacking. Somatic mutations represent a crucial source of genetic diversity for breeding and are especially remarkable in heterozygous perennial and asexual crops. In this study, we focused on a diploid sweet orange (Citrus sinensis) and constructed a haplotype-resolved genome using high fidelity (HiFi) reads, which revealed 10.6% new sequences. Based on the phased genome, we elucidate significant genetic admixtures and haplotype differences. We developed a somatic detection strategy that reveals hidden somatic mutations overlooked in a single reference genome. We generated a phased somatic variation map by combining high-depth whole-genome sequencing (WGS) data from 87 sweet orange somatic varieties. Notably, we found twice as many somatic mutations relative to a single reference genome. Using these hidden somatic mutations, we separated sweet oranges into seven major clades and provide insight into unprecedented genetic mosaicism and strong positive selection. Furthermore, these phased genomics data indicate that genomic heterozygous variations contribute to allele-specific expression during fruit development. By integrating allelic expression differences and somatic mutations, we identified a somatic mutation that induces increases in fruit size. Applications of phased genomics will lead to powerful approaches for discovering genetic variations and uncovering their effects in highly heterozygous plants. Our data provide insight into the hidden somatic mutation landscape in the sweet orange genome, which will facilitate citrus breeding.

Transposable elements cause the loss of self-incompatibility in citrus.

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.

PSEUDO-ETIOLATION IN LIGHT proteins reduce greening by binding GLK transcription factors.

Knocking out genes encoding proteins that downregulate the accumulation of pigments may lead to increases in crop quality and yield. PSEUDO-ETIOLATION IN LIGHT 1 (PEL1) downregulates the accumulation of carotenoids in carrot and chlorophyll in Arabidopsis and rice and may inhibit GOLDEN 2-LIKE (GLK) transcription factors. PEL1 belongs to a previously unstudied gene family found only in plants. We used CRISPR/Cas9 technology to knock out each member of the 4-member PEL gene family and both GLK genes in Arabidopsis. In pel mutants, chlorophyll levels were elevated in seedlings; after flowering, chloroplasts increased in size, and anthocyanin levels increased. Although the chlorophyll-deficient phenotype of glk1 glk2 was epistatic to pel1 pel2 pel3 pel4 in most of our experiments, glk1 glk2 was not epistatic to pel1 pel2 pel3 pel4 for the accumulation of anthocyanins in most of our experiments. The pel alleles attenuated growth, altered the accumulation of nutrients in seeds, disrupted an abscisic acid-inducible inhibition of seedling growth response that promotes drought tolerance, and affected the expression of genes associated with diverse biological functions, such as stress responses, cell wall metabolism hormone responses, signaling, growth, and the accumulation of phenylpropanoids and pigments. We found that PEL proteins specifically bind 6 transcription factors that influence the accumulation of anthocyanins, GLK2, and the carboxy termini of GLK1 and Arabidopsis thaliana myeloblastosis oncogene homolog 4 (AtMYB4). Our data indicate that the PEL proteins influence the accumulation of chlorophyll and many other processes, possibly by inhibiting GLK transcription factors and via other mechanisms, and that multiple mechanisms downregulate chlorophyll content.

Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non-photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty-four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG-localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi-feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops.

Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

Genomic conservation of crop wild relatives: A case study of citrus.

Conservation of crop wild relatives is critical for plant breeding and food security. The lack of clarity on the genetic factors that lead to endangered status or extinction create difficulties when attempting to develop concrete recommendations for conserving a citrus wild relative: the wild relatives of crops. Here, we evaluate the conservation of wild kumquat (Fortunella hindsii) using genomic, geographical, environmental, and phenotypic data, and forward simulations. Genome resequencing data from 73 accessions from the Fortunella genus were combined to investigate population structure, demography, inbreeding, introgression, and genetic load. Population structure was correlated with reproductive type (i.e., sexual and apomictic) and with a significant differentiation within the sexually reproducing population. The effective population size for one of the sexually reproducing subpopulations has recently declined to ~1,000, resulting in high levels of inbreeding. In particular, we found that 58% of the ecological niche overlapped between wild and cultivated populations and that there was extensive introgression into wild samples from cultivated populations. Interestingly, the introgression pattern and accumulation of genetic load may be influenced by the type of reproduction. In wild apomictic samples, the introgressed regions were primarily heterozygous, and genome-wide deleterious variants were hidden in the heterozygous state. In contrast, wild sexually reproducing samples carried a higher recessive deleterious burden. Furthermore, we also found that sexually reproducing samples were self-incompatible, which prevented the reduction of genetic diversity by selfing. Our population genomic analyses provide specific recommendations for distinct reproductive types and monitoring during conservation. This study highlights the genomic landscape of a wild relative of citrus and provides recommendations for the conservation of crop wild relatives.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Slawiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

SlBBX20 attenuates JA signalling and regulates resistance to Botrytis cinerea by inhibiting SlMED25 in tomato.

Jasmonic acid (JA) plays an important role in regulating plant growth and defence responses. Here, we show that a transcription factor that belongs to the B-box (BBX) family named SlBBX20 regulates resistance to Botrytis cinerea in tomato by modulating JA signalling. The response to JA was significantly suppressed when SlBBX20 was overexpressed in tomato. By contrast, the JA response was enhanced in SlBBX20 knockout lines. RNA sequencing analysis provided more evidence that SlBBX20 modulates the expression of genes that are involved in JA signalling. We found that SlBBX20 interacts with SlMED25, a subunit of the Mediator transcriptional co-activator complex, and prevents the accumulation of the SlMED25 protein and transcription of JA-responsive genes. JA contributes to the defence response against necrotrophic pathogens. Knocking out SlBBX20 or overexpressing SlMED25 enhanced tomato resistance to B. cinerea. The resistance was impaired when SlBBX20 was overexpressed in plants that also overexpressed SlMED25. These data show that SlBBX20 attenuates JA signalling by regulating SlMED25. Interestingly, in addition to developing enhanced resistance to B. cinerea, SlBBX20-KO plants also produced higher fruit yields. SlBBX20 is a potential target gene for efforts that aim to develop elite crop varieties using gene editing technologies.

Jasmonate activates a CsMPK6-CsMYC2 module that regulates the expression of ß-citraurin biosynthetic genes and fruit coloration in orange (Citrus sinensis).

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid ß-citraurin is responsible for the peel color in "Newhall" orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on ß-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and ß-citraurin production in "Newhall" orange. A MeJA treatment induced the expression of CsMYC2, which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for ß-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene ß-cyclase (CsLCYb), and ß-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce ß-citraurin biosynthesis in citrus by activating a CsMPK6-CsMYC2 cascade, thereby affecting fruit coloration.

Risk Evaluation of Benzovindiflupyr Resistance of Verticillium dahliae Population in Maine.

Verticillium dahliae causes Verticillium wilt, resulting in significant losses to potato production. Benzovindiflupyr, a succinate dehydrogenase inhibitor, effectively controls V. dahliae. However, frequent applications of the chemical may expedite the development of fungicide resistance in the pathogen population. To evaluate the risk of benzovindiflupyr resistance, 38 V. dahliae strains were obtained from diseased potatoes in Maine. The sensitivity of the field population was determined based on effective concentration for 50% inhibition (EC50), which ranged from 0.07 to 11.28 µg ml-1 with a median of 1.08. Segregated clusters of EC50 values indicated that Maine V. dahliae populations have developed benzovindiflupyr resistance. By exposing conidia of V. dahliae to a high concentration of benzovindiflupyr, 18 benzovindiflupyr-resistant mutants were obtained. To examine their fitness, the mutants were continuously subculture-transferred for up to 10 generations. Mycelial growth, conidial production, competitiveness, pathogenicity, and cross resistance of the 10th generation mutants were examined. Results showed that 50% of the resistant mutants retained an adaptive level in mycelial growth, and 60% maintained conidial production similar to their parents. Pathogenicity did not change for any of the mutants. No cross resistance was detected between benzovindiflupyr and either azoxystrobin, boscalid, fluopyram, or pyrimethanil. Thus, the resistance risk in V. dahliae to benzovindiflupyr should be considered in Maine potato production.

Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification.

Although interactions between the cytoplasmic and nuclear genomes occurred during diversification of many plants, the evolutionary conflicts due to cytonuclear interactions are poorly understood in crop breeding. Here, we constructed a pan-mitogenome and identified chimeric open reading frames (ORFs) generated by extensive structural variations (SVs). Meanwhile, short reads from 184 accessions of citrus species were combined to construct three variation maps for the nuclear, mitochondrial, and chloroplast genomes. The population genomic data showed discordant topologies between the cytoplasmic and nuclear genomes because of differences in mutation rates and levels of heteroplasmy from paternal leakage. An analysis of species-specific SVs indicated that mitochondrial heteroplasmy was common and that chloroplast heteroplasmy was undetectable. Interestingly, we found a prominent divergence in the mitogenomes and the highest genetic load in the, which may provide the basis for cytoplasmic male sterility (CMS) and thus influence the reshuffling of the cytoplasmic and nuclear genomes during hybridization. Using cytoplasmic replacement experiments, we identified a type of species-specific CMS in mandarin related to two chimeric mitochondrial genes. Our analyses indicate that cytoplasmic genomes from mandarin have rarely been maintained in hybrids and that paternal leakage produced very low levels of mitochondrial heteroplasmy in mandarin. A genome-wide association study (GWAS) provided evidence for three nuclear genes that encode pentatricopeptide repeat (PPR) proteins contributing to the cytonuclear interactions in the Citrus genus. Our study demonstrates the occurrence of evolutionary conflicts between cytoplasmic and nuclear genomes in citrus and has important implications for genetics and breeding.

Variation in the fruit development gene POINTED TIP regulates protuberance of tomato fruit tip.

The domestication of tomato has led to striking variations in fruit morphology. Here, we show a genome-wide association study (GWAS) to understand the development of the fruit tip and describe a POINTED TIP (PT) gene that encodes a C2H2-type zinc finger transcription factor. A single nucleotide polymorphism is found to change a histidine (H) to an arginine (R) in the C2H2 domain of PT and the two alleles are referred to as PTH and PTR. Knocking out PTH leads to development of pointed tip fruit. PTH functions to suppress pointed tip formation by downregulating the transcription of FRUTFULL 2 (FUL2), which alters the auxin transport. Our evolutionary analysis and previous studies by others suggest that the PTR allele likely hitch-hiked along with other selected loci during the domestication process. This study uncovers variation in PT and molecular mechanism underlying fruit tip development in tomato.

The transcription factors DcHB30 and DcWRKY75 antagonistically regulate ethylene-induced petal senescence in carnation (Dianthus caryophyllus).

Although numerous transcription factors with antagonistic activities have been shown to contribute to growth and development, whether and how they regulate senescence in plants is largely unknown. In this study, we investigated the role of antagonistic transcription factors in petal senescence in carnation (Dianthus caryophyllus), one of the most common types of ethylene-sensitive cut flowers produced worldwide. We identified DcHB30 that encodes a ZF-HD transcription factor that is down-regulated in ethylene-treated petal transcriptomes. We found that silencing DcHB30 accelerated ethylene-induced petal senescence and that DcHB30 physically interacts with DcWRKY75, a positive regulator of ethylene-induced petal senescence. Phenotypic characterization and molecular evidence indicated that DcHB30 and DcWRKY75 competitively regulate the expression of their co-targeted genes DcACS1, DcACO1, DcSAG12, and DcSAG29 by reciprocally inhibiting the DNA-binding activity of each other on the gene promoters. This transcriptional regulation mechanism demonstrates that these transcription factors serve as positive and negative regulators in ethylene-induced petal senescence in carnation. Thus, our study provides insights into how antagonizing transcription factors regulate plant senescence.

A novel regulatory complex mediated by Lanata (Ln) controls multicellular trichome formation in tomato.

Trichomes that originate from plant aerial epidermis act as mechanical and chemical barriers against herbivores. Although several regulators have recently been identified, the regulatory pathway underlying multicellular trichome formation remains largely unknown in tomato. Here, we report a novel HD-ZIP IV transcription factor, Lanata (Ln), a missense mutation which caused the hairy phenotype. Biochemical analyses demonstrate that Ln separately interacts with two trichome regulators, Woolly (Wo) and Hair (H). Genetic and molecular evidence demonstrates that Ln directly regulates the expression of H. The interaction between Ln and Wo can increase trichome density by enhancing the expression of SlCycB2 and SlCycB3, which we previously showed are involved in tomato trichome formation. Furthermore, SlCycB2 represses the transactivation of the SlCycB3 gene by Ln and vice versa. Our findings provide new insights into the novel regulatory network controlling multicellular trichome formation in tomato.

Heme oxygenase-independent bilin biosynthesis revealed by a hmox1 suppressor screening in Chlamydomonas reinhardtii.

Bilins are open-chain tetrapyrroles synthesized in phototrophs by successive enzymic reactions catalyzed by heme oxygenases (HMOXs/HOs) and ferredoxin-dependent biliverdin reductases (FDBRs) that typically serve as chromophore cofactors for phytochromes and phycobiliproteins. Chlamydomonas reinhardtii lacks both phycobiliproteins and phytochromes. Nonetheless, the activity and stability of photosystem I (PSI) and the catalytic subunit of magnesium chelatase (MgCh) named CHLH1 are significantly reduced and phototropic growth is significantly attenuated in a hmox1 mutant that is deficient in bilin biosynthesis. Consistent with these findings, previous studies on hmox1 uncovered an essential role for bilins in chloroplast retrograde signaling, maintenance of a functional photosynthetic apparatus, and the direct regulation of chlorophyll biosynthesis. In this study, we generated and screened a collection of insertional mutants in a hmox1 genetic background for suppressor mutants with phototropic growth restored to rates observed in wild-type 4A+ C. reinhardtii cells. Here, we characterized a suppressor of hmox1 named ho1su1 with phototrophic growth rates and levels of CHLH1 and PSI proteins similar to 4A+. Tetrad analysis indicated that a plasmid insertion co-segregated with the suppressor phenotype of ho1su1. Results from TAIL-PCR and plasmid rescue experiments demonstrated that the plasmid insertion was located in exon 1 of the HMOX1 locus. Heterologous expression of the bilin-binding reporter Nostoc punctiforme NpF2164g5 in the chloroplast of ho1su1 indicated that bilin accumulated in the chloroplast of ho1su1 despite the absence of the HMOX1 protein. Collectively, our study reveals the presence of an alternative bilin biosynthetic pathway independent of HMOX1 in the chloroplasts of Chlamydomonas cells.

Loss-of-function of SAWTOOTH 1 affects leaf dorsiventrality genes to promote leafy heads in lettuce.

The mechanisms underlying leafy heads in vegetables are poorly understood. Here, we cloned a quantitative trait locus (QTL) controlling leafy heads in lettuce (Lactuca sativa). The QTL encodes a transcription factor, SAWTOOTH 1 (LsSAW1), which has a BEL1-like homeodomain and is a homolog of Arabidopsis thaliana. A 1-bp deletion in Lssaw1 contributes to the development of leafy heads. Laser-capture microdissection and RNA-sequencing showed that LsSAW1 regulates leaf dorsiventrality and loss-of-function of Lssaw1 downregulates the expression of many adaxial genes but upregulates abaxial genes. LsSAW1 binds to the promoter region of the adaxial gene ASYMMETRIC LEAVES 1 (LsAS1) to upregulate its expression. Overexpression of LsAS1 compromised the effects of Lssaw1 on heading. LsSAW1 also binds to the promoter region of the abaxial gene YABBY 1 (LsYAB1), but downregulates its expression. Overexpression of LsYAB1 led to bending leaves in LsSAW1 genotypes. LsSAW1 directly interacts with KNOTTED 1 (LsKN1), which is necessary for leafy heads in lettuce. RNA-seq data showed that LsSAW1 and LsKN1 exert antagonistic effects on the expression of thousands of genes. LsSAW1 compromises the ability of LsKN1 to repress LsAS1. Our results suggest that downregulation or loss-of-function of adaxial genes and upregulation of abaxial genes allow for the development of leafy heads.