A truncated B-box zinc finger transcription factor confers drought sensitivity in modern cultivated tomatoes.

Enhancing drought tolerance in crops and understanding the underlying mechanisms have been subject of intense research. The precise function and molecular mechanisms of B-box zinc finger proteins (BBX) remain elusive. Here, we report a natural allele of BBX18 (BBX18TT) that encodes a C-terminal truncated protein. While most wild tomato germplasms contain the BBX18CC allele and show more drought tolerant, modern cultivated tomatoes mostly carry BBX18TT allele and are more drought sensitive. Knockout of BBX18 leads to improved drought tolerance in transgenic plants of cultivated tomato. Ascorbate peroxidase 1 (APX1) is identified as a BBX18-interacting protein that acts as a positive regulator of drought resistance in tomato. Chromatin immunoprecipitation sequencing analyses reveal that BBX18 binds to a unique cis-acting element of the APX1 promoter and represses its gene expression. This study provides insights into the molecular mechanism underlying drought resistance mediated by the BBX18-APX1 module in plants.

Nuclear factor Y-A3b binds to the SINGLE FLOWER TRUSS promoter and regulates flowering time in tomato.

The control of flowering time is essential for reproductive success and has a major effect on seed and fruit yield and other important agricultural traits in crops. Nuclear factors Y (NF-Ys) are transcription factors that form heterotrimeric protein complexes to regulate gene expression required for diverse biological processes, including flowering time control in plants. However, to our knowledge, there has been no report on mutants of individual NF-YA subunits that promote early flowering phenotype in plants. In this study, we identified SlNF-YA3b, encoding a member of the NF-Y transcription factor family, as a key gene regulating flowering time in tomato. Knockout of NF-YA3b resulted in an early flowering phenotype in tomato, whereas overexpression of NF-YA3b delayed flowering in transgenic tomato plants. NF-YA3b was demonstrated to form heterotrimeric protein complexes with multiple NF-YB/NF-YC heterodimers in yeast three-hybrid assays. Biochemical evidence indicated that NF-YA3b directly binds to the CCAAT cis-elements of the SINGLE FLOWER TRUSS (SFT) promoter to suppress its gene expression. These findings uncovered a critical role of NF-YA3b in regulating flowering time in tomato and could be applied to the management of flowering time in crops.

Defective mutations in STAY-GREEN 1, PHYTOENE SYNTHASE 1, and MYB12 genes lead to formation of green ripe fruit in tomato.

Modern tomatoes produce colorful mature fruits, but many wild tomato ancestors form green or gray green ripe fruits. Here, tomato cultivar 'Lvbaoshi' (LBS) that produces green ripe fruits was found to contain three recessive loci responsible for fruit development. The colorless peel of LBS fruits was caused by a 603 bp deletion in the promoter of SlMYB12. The candidate genes of the remaining two loci were identified as STAY-GREEN 1 (SlSGR1) and PHYTOENE SYNTHASE 1 (SlPSY1). SGR1 and PSY1 co-suppression by RNAi converted the pink fruits into green ripe fruits in transgenic plants. An amino acid change in PSY1 and a deletion in the promoter of SGR1 were also identified in several wild tomatoes bearing green or gray ripe fruits. Overexpression of PSY1 from green ripe fruit wild tomatoes in LBS plants could only partially rescue the green ripe fruit phenotype of LBS, and transgenic lines expressing ProSGR1::SGR1 from Solanum pennellii also failed to convert purple-flesh into red-flesh fruits. This work uncovers a novel regulatory mechanism by which SlMYB12, SlPSY1, and SlSGR1 control fruit color in cultivated and some wild tomato species.

EARLY FLOWERING is a dominant gain-of-function allele of FANTASTIC FOUR 1/2c that promotes early flowering in tomato.

Flowering time, an important factor in plant adaptability and genetic improvement, is regulated by various genes in tomato (Solanum lycopersicum). In this study, we characterized a tomato mutant, EARLY FLOWERING (EF), that developed flowers much earlier than its parental control. EF is a dominant gain-of-function allele with a T-DNA inserted 139 bp downstream of the stop codon of FANTASTIC FOUR 1/2c (FAF1/2c). The transcript of SlFAF1/2c was at elevated levels in the EF mutant. Overexpressing SlFAF1/2c in tomato plants phenocopied the early flowering trait of the EF mutant. Knocking out SlFAF1/2c in the EF mutant reverted the early flowering phenotype of the mutant to the normal flowering time of the wild-type tomato plants. SlFAF1/2c promoted the floral transition by shortening the vegetative phase rather than by reducing the number of leaves produced before the emergence of the first inflorescence. The COP9 signalosome subunit 5B (CSN5B) was shown to interact with FAF1/2c, and knocking out CSN5B led to an early flowering phenotype in tomato. Interestingly, FAF1/2c was found to reduce the accumulation of the CSN5B protein by reducing its protein stability. These findings imply that FAF1/2c regulates flowering time in tomato by reducing the accumulation and stability of CSN5B, which influences the expression of SINGLE FLOWER TRUSS (SFT), JOINTLESS (J) and UNIFLORA (UF). Thus, a new allele of SlFAF1/2c was discovered and found to regulate flowering time in tomato.

The chromosome-scale reference genome and transcriptome analysis of Solanum torvum provides insights into resistance to root-knot nematodes.

Solanum torvum (Swartz) (2n = 24) is a wild Solanaceae plant with high economic value that is used as a rootstock in grafting for Solanaceae plants to improve the resistance to a soil-borne disease caused by root-knot nematodes (RKNs). However, the lack of a high-quality reference genome of S. torvum hinders research on the genetic basis for disease resistance and application in horticulture. Herein, we present a chromosome-level assembly of genomic sequences for S. torvum combining PacBio long reads (HiFi reads), Illumina short reads and Hi-C scaffolding technology. The assembled genome size is ~1.25 Gb with a contig N50 and scaffold N50 of 38.65 Mb and 103.02 Mb, respectively as well as a BUSCO estimate of 98%. GO enrichment and KEGG pathway analysis of the unique S. torvum genes, including NLR and ABC transporters, revealed that they were involved in disease resistance processes. RNA-seq data also confirmed that 48 NLR genes were highly expressed in roots and fibrous roots and that three homologous NLR genes (Sto0288260.1, Sto0201960.1 and Sto0265490.1) in S. torvum were significantly upregulated after RKN infection. Two ABC transporters, ABCB9 and ABCB11 were identified as the hub genes in response to RKN infection. The chromosome-scale reference genome of the S. torvum will provide insights into RKN resistance.

SlTCP24 and SlTCP29 synergistically regulate compound leaf development through interacting with SlAS2 and activating transcription of SlCKX2 in tomato.

The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question. SlTCP24 and SlTCP29, two plant-specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29. SlTCP24 and SlTCP29 could bind to the TCP-binding cis-element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double-gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin. This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study.

SlGH3.15, a member of the GH3 gene family, regulates lateral root development and gravitropism response by modulating auxin homeostasis in tomato.

Multiple Gretchen Hagen 3 (GH3) genes have been implicated in a range of processes in plant growth and development through their roles in maintaining hormonal homeostasis. However, there has only been limited study on the functions of GH3 genes in tomato (Solanum lycopersicum). In this work, we investigated the important function of SlGH3.15, a member of the GH3 gene family in tomato. Overexpression of SlGH3.15 led to severe dwarfism in both the above- and below-ground sections of the plant, accompanied by a substantial decrease in free IAA content and reduction in the expression of SlGH3.9, a paralog of SlGH3.15. Exogenous supply of IAA negatively affected the elongation of the primary root and partially restored the gravitropism defects in SlGH3.15-overexpression lines. While no phenotypic change was observed in the SlGH3.15 RNAi lines, double knockout lines of SlGH3.15 and SlGH3.9 were less sensitive to treatments with the auxin polar transport inhibitor. Overall, these findings revealed important roles of SlGH3.15 in IAA homeostasis and as a negative regulator of free IAA accumulation and lateral root formation in tomato.

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 tomato CONSTANS-LIKE protein SlCOL1 regulates fruit yield by repressing SFT gene expression.

BACKGROUND: CONSTANS (CO) and CONSTANS-LIKE (COL) transcription factors have been known to regulate a series of cellular processes including the transition from the vegetative growth to flower development in plants. However, their role in regulating fruit yield in tomato is poorly understood. RESULT: In this study, the tomato ortholog of Arabidopsis CONSTANS, SlCOL1, was shown to play key roles in the control of flower development and fruit yield. Suppression of SlCOL1 expression in tomato was found to lead to promotion of flower and fruit development, resulting in increased tomato fruit yield. On the contrary, overexpression of SlCOL1 disturbed flower and fruit development, and significantly reduced tomato fruit yield. Genetic and biochemical evidence indicated that SlCOL1 controls inflorescence development by directly binding to the promoter region of tomato inflorescence-associated gene SINGLE-FLOWER TRUSS (SFT) and negatively regulating its expression. Additionally, we found that SlCOL1 can also negatively regulate fruit size in tomato. CONCLUSIONS: Tomato SlCOL1 binds to the promoter of the SFT gene, down-regulates its expression, and plays a key role in reducing the fruit size.

Regulation of tomato fruit elongation by transcription factor BZR1.7 through promotion of SUN gene expression.

Fruit shape is an important biological trait that is also of special commercial value in tomato. The SUN gene has been known as a key regulator of tomato fruit elongation for years, but the molecular mechanisms underlying its transcriptional regulation remain little understood. Here, a unique BZR1-like transcription factor, BZR1.7, was identified as a trans-acting factor of the SUN gene promoter that bound to the conserved E-box of the promoter to promote SUN gene expression. Overexpression of BZR1.7 in tomato led to elevated SUN gene expression and formation of elongated fruits. Plants of the BZR1.7 knockout mutant created by gene editing did not exhibit an observable fruit shape phenotype, suggesting possible functional redundancy of BZR1-like genes in tomato. There were seven BZR1-like genes in the tomato genome and overexpression of BZR1.5 and BZR1.6 led to elongated fruit phenotypes similar to those observed in the BZR1.7 overexpression lines, further supporting the notion of functional redundancy of BZR1-like genes in tomato fruit shape specification. Microscopic analysis revealed that there was a decreased number of cell layers in the fruit pericarp in the BZR1.7 overexpression lines. These findings offer new insights into the regulatory mechanism by which BZR1.7 promotes SUN gene expression and regulates fruit elongation in tomato.

KOMPEITO, an Atypical Arabidopsis Rhomboid-Related Gene, Is Required for Callose Accumulation and Pollen Wall Development.

Fertilization is a key event for sexually reproducing plants. Pollen-stigma adhesion, which is the first step in male-female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a ß-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen-stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.

Tomato methionine sulfoxide reductase B2 functions in drought tolerance by promoting ROS scavenging and chlorophyll accumulation through interaction with Catalase 2 and RBCS3B.

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of common metabolism and as the result of defense and development. ROS readily oxidizes methionine (Met) residues of proteins to form Met-R-sulfoxide or Met-S-sulfoxide (MetSO), resulting in protein inactivation or malfunction. Although it is known that MetSO can be reverted to Met by methionine sulfoxide reductase (Msr), the mechanism how Msr interacts with its target proteins is poorly understood. In this study, two target proteins of tomato MsrB2 (SlMsrB2), catalase 2 (CAT2) and the Rubisco small subunit RBCS3B, were identified. Silencing of SlMsrB2 by RNA interference (RNAi) in tomato led to decreased drought tolerance, accompanied by increased ROS accumulation and chlorophyll degradation. By contrast, overexpression of SlMsrB2 in tomato significantly reduced ROS accumulation and enhanced drought tolerance. Protein interaction analysis showed that SlMsrB2 interacts with CAT2 and RBCS3B in vitro and in planta. Silencing of CAT2 by RNAi and RBCS3B by virus-induced gene silencing (VIGS) resulted in development of pale green leaves and enhanced ROS accumulation in tomato plants. These results demonstrate that SlMsrB2 functions in drought tolerance and promotes chlorophyll accumulation by modulating ROS accumulation.

Genetic Loci Underlying Awn Morphology in Barley.

Barley awns are highly active in photosynthesis and account for 30-50% of grain weight in barley. They are diverse in length, ranging from long to awnless, and in shape from straight to hooded or crooked. Their diversity and importance have intrigued geneticists for several decades. A large collection of awnness mutants are available-over a dozen of them have been mapped on chromosomes and a few recently cloned. Different awnness genes interact with each other to produce diverse awn phenotypes. With the availability of the sequenced barley genome and application of new mapping and gene cloning strategies, it will now be possible to identify and clone more awnness genes. A better understanding of the genetic basis of awn diversity will greatly facilitate development of new barley cultivars with improved yield, adaptability and sustainability.

Genetic Interactions of Awnness Genes in Barley.

Awns are extending structures from lemmas in grasses and are very active in photosynthesis, contributing directly to the filling of the developing grain. Barley (Hordeum vulgare L.) awns are highly diverse in shape and length and are known to be controlled by multiple awn-related genes. The genetic effects of these genes on awn diversity and development in barley are multiplexed and include complementary effect, cumulative effect, duplicate effect, recessive epistasis, dominant epistasis, and inhibiting effect, each giving a unique modified Mendelian ratio of segregation. The complexity of gene interactions contributes to the awn diversity in barley. Excessive gene interactions create a challenging task for genetic mapping and specific strategies have to be developed for mapping genes with specific interactive effects. Awn gene interactions can occur at different levels of gene expression, from the transcription factor-mediated gene transcription to the regulation of enzymes and metabolic pathways. A better understanding of gene interactions will greatly facilitate deciphering the genetic mechanisms underlying barley awn diversity and development.

Genetic analysis reveals four interacting loci underlying awn trait diversity in barley (Hordeum vulgare).

Barley (Hordeum vulgare) awns contribute to grain yield, but the genetic basis of awn development remains largely unclear. Five barley lines differing in awn traits and row types were used to create four F2 populations. Genetic analyses revealed that four pairs of genes were involved in awn development: A/a (awnless/awned), B/b (awnless/awned), H/h (hooded/straight), and L/l (long/short). Of these four loci, A, H and L functioned on both central rows (CR) and lateral rows (LR) of the barley spikes, while B exhibited effect only on LR. A and B had duplicate effects on LR, and both showed dominant epistasis to loci H and L, whereas H was epistatic to L. Meanwhile, A and B were found to be genetically linked, with a row-type locus V located between them. The genetic distances of A-V and B-V were estimated to be 9.6 and 7.7 cM, respectively. Literature search suggested that A, H and V may correspond to the reported Lks1, Kap1 and Vrs1, respectively, whereas B is a novel gene specifically controlling awn development on LR, designated as Lsa1 for lateral spikelet awnless 1. The only barley homolog of wheat awn inhibitor gene B1, HORVU2Hr1G077570, is a potential candidate of Lsa1.

The Lotus japonicus Ubiquitin Ligase SIE3 Interacts With the Transcription Factor SIP1 and Forms a Homodimer.

The symbiosis receptor kinase SymRK plays an essential role in symbiotic signal transduction and nodule organogenesis. Several proteins bind to SymRK, but how the symbiosis signals are transduced from SymRK to downstream components remains elusive. We previously demonstrated that both SymRK interacting protein 1 (SIP1, an ARID-type DNA-binding protein) and SymRK interacting E3 ligase [SIE3, a RING (Really Interesting New Gene)-containing E3 ligase] interact with SymRK to regulate downstream cellular responses in Lotus japonicus during the legume-rhizobia symbiosis. Here, we show that SIE3 interacts with SIP1 in both yeast cells and Nicotiana benthamiana. SIE3 associated with itself and formed a homodimer. The cysteine 266 residue was found to be essential for SIE3 dimerization and for promoting nodulation in transgenic hairy roots of L. japonicus. Our findings provide a foundation for further investigating the regulatory mechanisms of the SymRK-mediated signaling pathway, as well as the biological function of E3 ligase dimerization in nodule organogenesis.

A missense mutation in Large Grain Size 1 increases grain size and enhances cold tolerance in rice.

Grain shape is controlled by quantitative trait loci (QTLs) in rice (Oryza sativa L.). A rice mutant (JF178) with long and large grains has been used in a breeding program for over a decade, but its genetic basis has been unclear. Here, a semi-dominant QTL, designated Large Grain Size 1 (LGS1), was cloned and the potential molecular mechanism of LGS1 function was studied. Near-isogenic lines (NILs) and a map-based approach were employed to clone the LGS1 locus. LGS1 encodes the OsGRF4 transcription factor and contains a 2 bp missense mutation in the coding region that coincides with the putative pairing site of miRNA396. The LGS1 transcript levels in the mutant line were found to be higher than the lgs1 transcript levels in the control plants, suggesting that the mutation might disrupt the pairing of the LGS1 mRNA with miR396. In addition to producing larger grains, LGS1 also enhanced cold tolerance at the seedling stage and increased the survival rate of seedlings after cold stress treatment. These findings indicate that the mutation in LGS1 appears to disturb the GRF4-miR396 stress response network and results in the development of enlarged grains and enhancement of cold tolerance in rice.

An MAP kinase interacts with LHK1 and regulates nodule organogenesis in Lotus japonicus.

Symbiosis receptor-like kinase (SymRK) is a key protein mediating the legume-Rhizobium symbiosis. Our previous work has identified an MAP kinase kinase, SIP2, as a SymRK-interacting protein to positively regulate nodule organogenesis in Lotus japonicus, suggesting that an MAPK cascade might be involved in Rhizobium-legume symbiosis. In this study, LjMPK6 was identified as a phosphorylation target of SIP2. Stable transgenic L. japonicus with RNAi silencing of LjMPK6 decreased the numbers of nodule primordia (NP) and nodule, while plants overexpressing LjMPK6 increased the numbers of nodule, infection threads (ITs), and NP, indicating that LjMPK6 plays a positive role in nodulation. LjMPK6 could interact with a cytokinin receptor, LHK1 both in vivo and in vitro. LjMPK6 was shown to compete with LHP1 to bind to the receiver domain (RD) of LHK1and to downregulate the expression of two LjACS (1-aminocyclopropane-1-carboxylic acid synthase) genes and ethylene levels during nodulation. This study demonstrated an important role of LjMPK6 in regulation of nodule organogenesis and ethylene production in L. japonicus.

Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) regulates cell cycling potentially by cooperating with nucleosome assembly protein AtNAP1;1.

BACKGROUND: In mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown. RESULTS: Using nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell  cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction. CONCLUSIONS: As a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation through the cooperation of a GTP-binding protein with a nucleosome assembly/disassembly protein in Arabidopsis.

Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9.

The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.