Metabolic Engineering of Escherichia coli for the Biosynthesis of 3-Phenylpropionic Acid and 3-Phenylpropyl Acetate.

3-Phenylpropionic acid (3PPA) and its derivative 3-phenylpropyl acetate (3PPAAc) are important aromatic compounds with broad applications in the cosmetics and food industries. In this study, we constructed a plasmid-free 3PPA-producing Escherichia coli strain and designed a novel 3PPAAc biosynthetic pathway. A module containing tyrosine ammonia lyase and enoate reductase, evaluated under the control of different promoters, was combined with phenylalanine-overproducing strain E. coli ATCC31884, enabling the plasmid-free de novo production of 218.16 ± 43.62 mg L-1 3PPA. The feasibility of the pathway was proved by screening four heterologous alcohol acetyltransferases, which catalyzed the transformation of 3-phenylpropyl alcohol into 3PPAAc. Afterward, 94.59 ± 16.25 mg L-1 3PPAAc was achieved in the engineered E. coli strain. Overall, we have not only demonstrated the potential of de novo synthesis of 3PPAAc in microbes for the first time but also provided a platform for the future of biosynthesis of other aromatic compounds.

The Arabidopsis thaliana trehalose-6-phosphate phosphatase gene AtTPPI regulates primary root growth and lateral root elongation.

Roots are the main organs through which plants absorb water and nutrients. As the key phytohormone involved in root growth, auxin functions in plant environmental responses by modulating auxin synthesis, distribution and polar transport. The Arabidopsis thaliana trehalose-6-phosphate phosphatase gene AtTPPI can improve root architecture, and tppi1 mutants have significantly shortened primary roots. However, the mechanism underlying the short roots of the tppi1 mutant and the upstream signaling pathway and downstream genes regulated by AtTPPI are unclear. Here, we demonstrated that the AtTPPI gene could promote auxin accumulation in AtTPPI-overexpressing plants. By comparing the transcriptomic data of tppi1 and wild-type roots, we found several upregulations of auxin-related genes, including GH3.3, GH3.9 and GH3.12, may play an important role in the AtTPPI gene-mediated auxin transport signaling pathway, ultimately leading to changes in auxin content and primary root length. Moreover, increased AtTPPI expression can regulate primary root growth and lateral root elongation under different concentration of nitrate conditions. Overall, constitutive expression of AtTPPI increased auxin contents and improved lateral root elongation, constituting a new method for improving the nitrogen utilization efficiency of plants.

SlGT11 controls floral organ patterning and floral determinacy in tomato.

BACKGROUND: Flower development directly affects fruit production in tomato. Despite the framework mediated by ABC genes have been established in Arabidopsis, the spatiotemporal precision of floral development in tomato has not been well examined. RESULTS: Here, we analyzed a novel tomato stamenless like flower (slf) mutant in which the development of stamens and carpels is disturbed, with carpelloid structure formed in the third whorl and ectopic formation of floral and shoot apical meristem in the fourth whorl. Using bulked segregant analysis (BSA), we assigned the causal mutation to the gene Solanum lycopersicum GT11 (SlGT11) that encodes a transcription factor belonging to Trihelix gene family. SlGT11 is expressed in the early stages of the flower and the expression becomes more specific to the primordium position corresponding to stamens and carpels in later stages of the floral development. Further RNAi silencing of SlGT11 verifies the defective phenotypes of the slf mutant. The carpelloid stamen in slf mutant indicates that SlGT11 is required for B-function activity in the third whorl. The failed termination of floral meristem and the occurrence of floral reversion in slf indicate that part of the C-function requires SlGT11 activity in the fourth whorl. Furthermore, we find that at higher temperature, the defects of slf mutant are substantially enhanced, with petals transformed into sepals, all stamens disappeared, and the frequency of ectopic shoot/floral meristem in fourth whorl increased, indicating that SlGT11 functions in the development of the three inner floral whorls. Consistent with the observed phenotypes, it was found that B, C and an E-type MADS-box genes were in part down regulated in slf mutants. CONCLUSIONS: Together with the spatiotemporal expression pattern, we suggest that SlGT11 functions in floral organ patterning and maintenance of floral determinacy in tomato.

AUREA maintains the balance between chlorophyll synthesis and adventitious root formation in tomato.

Flooding tolerance is an important trait for tomato breeding. In this study, we obtained a recessive mutant exhibiting highly enhanced submergence resistance. Phenotypical analyses showed that this resistant to flooding (rf) mutant displays slightly chlorotic leaves and spontaneous initiation of adventitious roots (ARs) on stems. The mutation was mapped to the phytochromobilin synthase gene AUREA (AU), in which a single amino acid substitution from asparagine to tyrosine occurred. In addition to the classic function of AU in phytochrome and chlorophyll biogenesis in leaves, we uncovered its novel role in mediating AR formation on stems. We further observed temporal coincidence of the two phenotypes in the rf mutant: chlorosis and spontaneous AR formation and revealed that AU functions by maintaining heme homeostasis. Interestingly, our grafting results suggest that heme might play roles in AR initiation via long-distance transport from leaves to stems. Our results present genetic evidence for the involvement of the AU-heme oxygenase-1-heme pathway in AR initiation in tomato. As fruit production and yield in the rf mutant are minimally impacted, the mutation identified in this study may provide a target for biotechnological renovation of tomato germplasm in future breeding.

IDD16 negatively regulates stomatal initiation via trans-repression of SPCH in Arabidopsis.

In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by SPEECHLESS (SPCH). Phosphorylation of SPCH at the post-translational level has been reported to regulate stomatal development. Here we report that IDD16 acts as a negative regulator for stomatal initiation by directly regulating SPCH transcription. In Arabidopsis, IDD16 overexpression decreased abaxial stomatal density in a dose-dependent manner. Time course analysis revealed that the initiation of stomatal precursor cells in the IDD16-OE plants was severely inhibited. Consistent with these findings, the transcription of SPCH was greatly repressed in the IDD16-OE plants. In contrast, IDD16-RNAi transgenic line resulted in enhanced stomatal density, suggesting that IDD16 is an intrinsic regulator of stomatal development. ChIP analysis indicated that IDD16 could directly bind to the SPCH promoter. Furthermore, Arabidopsis plants overexpressing IDD16 exhibited significantly increased drought tolerance and higher integrated water use efficiency (WUE) due to reduction in leaf transpiration. Collectively, our results established that IDD16 negatively regulates stomatal initiation via trans-repression of SPCH, and thus provide a practical tool for increasing plant WUE through the manipulation of IDD16 expression.

Loss or duplication of key regulatory genes coincides with environmental adaptation of the stomatal complex in Nymphaea colorata and Kalanchoe laxiflora.

The stomatal complex is critical for gas and water exchange between plants and the atmosphere. Originating over 400 million years ago, the structure of the stomata has evolved to facilitate the adaptation of plants to various environments. Although the molecular mechanism of stomatal development in Arabidopsis has been widely studied, the evolution of stomatal structure and its molecular regulators in different species remains to be answered. In this study, we examined stomatal development and the orthologues of Arabidopsis stomatal genes in a basal angiosperm plant, Nymphaea colorata, and a member of the eudicot CAM family, Kalanchoe laxiflora, which represent the adaptation to aquatic and drought environments, respectively. Our results showed that despite the conservation of core stomatal regulators, a number of critical genes were lost in the N. colorata genome, including EPF2, MPK6, and AP2C3 and the polarity regulators BASL and POLAR. Interestingly, this is coincident with the loss of asymmetric divisions during the stomatal development of N. colorata. In addition, we found that the guard cell in K. laxiflora is surrounded by three or four small subsidiary cells in adaxial leaf surfaces. This type of stomatal complex is formed via repeated asymmetric cell divisions and cell state transitions. This may result from the doubled or quadrupled key genes controlling stomatal development in K. laxiflora. Our results show that loss or duplication of key regulatory genes is associated with environmental adaptation of the stomatal complex.

Construction of a Functional Casparian Strip in Non-endodermal Lineages Is Orchestrated by Two Parallel Signaling Systems in Arabidopsis thaliana.

The Casparian strip in the root endodermis forms an apoplastic barrier between vascular tissues and outer ground tissues to enforce selective absorption of water and nutrients. Because of its cell-type specificity, the presence of a Casparian strip is used as a marker for a functional endodermis. Here, we examine the minimal regulators required for reprograming non-endodermal cells to build a functional Casparian strip. We demonstrate that the transcription factor SHORT-ROOT (SHR) serves as a master regulator and promotes Casparian strip formation through two independent activities: inducing the expression of essential Casparian strip enzymes via MYB36 and directing the subcellular localization of Casparian strip formation via SCARECROW (SCR). However, this hierarchical signaling cascade still needs SHR-independent small peptides, derived from the stele, to eventually build a functional Casparian strip in non-endodermal cells. Our study provides a synthetic approach to induce Casparian-strip-containing endodermis using a minimal network of regulators and reveals the deployment of both apoplastic and symplastic communication in the promotion of a specific cell fate.

The RING Finger E3 Ligase SpRing is a Positive Regulator of Salt Stress Signaling in Salt-Tolerant Wild Tomato Species.

Protein ubiquitination in plants plays critical roles in many biological processes, including adaptation to abiotic stresses. Previously, RING finger E3 ligase has been characterized during salt stress response in several plant species, but little is known about its function in tomato. Here, we report that SpRing, a stress-inducible gene, is involved in salt stress signaling in wild tomato species Solanum pimpinellifolium 'PI365967'. In vitro ubiquitination assay revealed that SpRing is an E3 ubiquitin ligase and the RING finger conserved region is required for its activity. SpRing is expressed in all tissues of wild tomato and up-regulated by salt, drought and osmotic stresses, but repressed by low temperature. Green fluorescent protein (GFP) fusion analysis showed that SpRing is localized at the endoplasmic reticulum. Silencing of SpRing through a virus-induced gene silencing approach led to increased sensitivity to salt stress in wild tomato. Overexpression of SpRing in Arabidopsis thaliana resulted in enhanced salt tolerance during seed germination and early seedling development. The expression levels of certain key stress-related genes are altered both in SpRing-overexpressing Arabidopsis plants and virus-induced gene silenced tomato seedlings. Taken together, our results indicate that SpRing is involved in salt stress and functions as a positive regulator of salt tolerance.