Lskipk Lsatpase double mutants are necessary and sufficient for the compact plant architecture of butterhead lettuce.

Lettuce, an important leafy vegetable crop worldwide, has rich variations in plant architecture. Butterhead lettuce, a popular horticultural type, has a unique plant architecture with loose leafy heads. The genetic and molecular mechanisms for such a compact plant architecture remain unclear. In this study we constructed a segregating population through crossing a butterhead cultivar and a stem lettuce cultivar. Genetic analysis identified the LsKIPK gene, which encodes a kinase, as the candidate gene controlling butterhead plant architecture. The Lskipk gene in the butterhead parent had a nonsense mutation, leading to a partial predicted protein. CRISPR/Cas9 and complementation tests verified its functions in plant architecture. We showed that the loss of function of LsKIPK is necessary but not sufficient for the butterhead plant architecture. To identify additional genes required for butterhead lettuce, we crossed a butterhead cultivar and a crisphead cultivar, both with the mutated Lskipk gene. Genetic mapping identified a new gene encoding an ATPase contributing to butterhead plant architecture. Knockout and complementation tests showed that loss of function of LsATPase is also required for the development of butterhead plant architecture. The Lskipk Lsatpase double mutation could reduce leaf size and leaf angle, leading to butterhead plant architecture. Expression and cytology analysis indicated that the loss of function of LsKIPK and LsATPase contributed to butterhead plant architecture by regulating cell wall development, a regulatory mechanism different from that for crisphead. This study provides new gene resources and theory for the breeding of the crop ideotype.

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.

Transcription factor SlWRKY50 enhances cold tolerance in tomato by activating the jasmonic acid signaling.

Tomato (Solanum lycopersicum) is a cold-sensitive crop but frequently experiences low-temperature stimuli. However, tomato responses to cold stress are still poorly understood. Our previous studies have shown that using wild tomato (Solanum habrochaites) as rootstock can significantly enhance the cold resistance of grafted seedlings, in which a high concentration of jasmonic acids (JAs) in scions exerts an important role, but the mechanism of JA accumulation remains unclear. Herein, we discovered that tomato SlWRKY50, a Group II WRKY transcription factor that is cold inducible, responds to cold stimuli and plays a key role in JA biosynthesis. SlWRKY50 directly bound to the promoter of tomato allene oxide synthase gene (SlAOS), and overexpressing SlWRKY50 improved tomato chilling resistance, which led to higher levels of Fv/Fm, antioxidative enzymes, SlAOS expression, and JA accumulation. SlWRKY50-silenced plants, however, exhibited an opposite trend. Moreover, diethyldithiocarbamate acid (a JA biosynthesis inhibitor) foliar treatment drastically reduced the cold tolerance of SlWRKY50-overexpression plants to wild-type levels. Importantly, SlMYC2, the key regulator of the JA signaling pathway, can control SlWRKY50 expression. Overall, our research indicates that SlWRKY50 promotes cold tolerance by controlling JA biosynthesis and that JA signaling mediates SlWRKY50 expression via transcriptional activation by SlMYC2. Thus, this contributes to the genetic knowledge necessary for developing cold-resistant tomato varieties.

The essential role of jasmonate signaling in Solanum habrochaites rootstock-mediated cold tolerance in tomato grafts.

Tomato (Solanum lycopersicum) is among the most important vegetables across the world, but cold stress usually affects its yield and quality. The wild tomato species Solanum habrochaites is commonly utilized as rootstock for enhancing resistance against abiotic stresses in cultivated tomato, especially cold resistance. However, the underlying molecular mechanism remains unclear. In this research, we confirmed that S. habrochaites rootstock can improve the cold tolerance of cultivated tomato scions, as revealed by growth, physiological, and biochemical indicators. Furthermore, transcriptome profiling indicated significant differences in the scion of homo- and heterografted seedlings, including substantial changes in jasmonic acid (JA) biosynthesis and signaling, which were validated by RT-qPCR analysis. S. habrochaites plants had a high basal level of jasmonate, and cold stress caused a greater amount of active JA-isoleucine in S. habrochaites heterografts. Moreover, exogenous JA enhanced while JA inhibitor decreased the cold tolerance of tomato grafts. The JA biosynthesis-defective mutant spr8 also showed increased sensitivity to cold stress. All of these results demonstrated the significance of JA in the cold tolerance of grafted tomato seedlings with S. habrochaites rootstock, suggesting a future direction for the characterization of the natural variation involved in S. habrochaites rootstock-mediated cold tolerance.

Systematic Analysis of the Grafting-Related Glucanase-Encoding GH9 Family Genes in Pepper, Tomato and Tobacco.

Grafting is an important agricultural practice to control soil-borne diseases, alleviate continuous cropping problems and improve stress tolerance in vegetable industry, but it is relatively less applied in pepper production. A recent study has revealed the key roles of ß-1, 4-glucanase in graft survival. We speculated that the GH9 family gene encoding glucanase may be involved in the obstacles of pepper grafting. Therefore, we performed a systematic analysis of the GH9 family in pepper, tomato and tobacco. A total of 25, 24 and 42 GH9 genes were identified from these three species. Compared with the orthologues of other solanaceous crops, the deduced pepper GH9B3 protein lacks a conserved motif (Motif 5). Promoter cis-element analysis revealed that a wound-responsive element exists in the promoter of tobacco NbGH9B3, but it is absent in the GH9B3 promoter of most solanaceous crops. The auxin-responsive related element is absent in CaGH9B3 promoter, but it presents in the promoter of tobacco, tomato, potato and petunia GH9B3. Tissue and induction expression profiles indicated that GH9 family genes are functionally differentiated. Nine GH9 genes, including CaGH9B3, were detected expressing in pepper stem. The expression patterns of NbGH9B3 and CaGH9B3 in grafting were different in our test condition, with obvious induction in tobacco but repression in pepper. Furthermore, weighted correlation network analysis (WGCNA) revealed 58 transcription factor genes highly co-expressed with NbGH9B3. Eight WRKY binding sites were detected in the promoter of NbGH9B3, and several NbWRKYs were highly co-expressed with NbGH9B3. In conclusion, the missing of Motif 5 in CaGH9B3, and lacking of wound- and auxin-responsive elements in the gene promoter are the potential causes of grafting-related problems in pepper. WRKY family transcription factors could be important regulator of NbGH9B3 in tobacco grafting. Our analysis points out the putative regulators of NbGH9B3, which would be helpful to the functional validation and the study of signal pathways related to grafting in the future.