Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis,plant development,and carotenoid metabolism in fruits

Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a‐overexpressing lines (OE‐CRY1a‐1 and OE‐CRY1a‐2). In comparison with wild‐type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE‐CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA‐seq, qRT‐PCR, and LC‐MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down‐regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE‐CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß‐carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.     

Emerging Roles and New Paradigms in Signaling Mechanisms of Plant Cryptochromes

Analogous to the opsin-based receptors in animals, plants contain a diverse and elaborate set of photoreceptors to perceive a much wider spectrum of light and adapt to varying light conditions. Cryptochromes (CRYs), the blue/UV-A light sensing receptors, represent one such class of photoreceptors found ubiquitously in plants. Although structurally similar to DNA photolyases which could repair UV-induced DNA damage, photoactivated CRYs, instead, initiate signal transduction pathways, which lead to gene expression changes and eventually more overt photomorphogenic responses. Apart from the well-established roles of CRYs in regulating seedling de-etiolation, flowering time, and circadian clock, recent reports have highlighted their roles in controlling other aspects of plant development as well. This review attempts to describe the novel/atypical roles of CRYs that have emerged in the past few years, and also present an account of the various signaling components involved in CRY signal transduction pathway.     

Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content

Cryptochromes are blue light photoreceptors found in plants, bacteria, and animals. In Arabidopsis, cryptochrome 2 (cry2) is involved primarily in the control of flowering time and in photomorphogenesis under low-fluence light. No data on the function of cry2 are available in plants, apart from Arabidopsis (Arabidopsis thaliana). Expression of the tomato (Solanum lycopersicum) CRY2 gene was altered through a combination of transgenic overexpression and virus-induced gene silencing. Tomato CRY2 overexpressors show phenotypes similar to but distinct from their Arabidopsis counterparts (hypocotyl and internode shortening under both low- and high-fluence blue light), but also several novel ones, including a high-pigment phenotype, resulting in overproduction of anthocyanins and chlorophyll in leaves and of flavonoids and lycopene in fruits. The accumulation of lycopene in fruits is accompanied by the decreased expression of lycopene beta-cyclase genes. CRY2 overexpression causes an unexpected delay in flowering, observed under both short- and long-day conditions, and an increased outgrowth of axillary branches. Virus-induced gene silencing of CRY2 results in a reversion of leaf anthocyanin accumulation, of internode shortening, and of late flowering in CRY2-overexpressing plants, whereas in wild-type plants it causes a minor internode elongation.     

Decreasing fructose‐1,6‐bisphosphate aldolase activity reduces plant growth and tolerance to chilling stress in tomato seedlings

In northern China, low temperature is the most common abiotic stresses for tomato plants cultivated in solar‐greenhouse in winter. We recently found that the expression and enzyme activity of fructose‐1,6‐bisphosphate aldolases (FBAs) in tomato, which are important enzymes in the Calvin–Benson cycle (CBC), were significantly altered in tomato seedlings subjected to heat/cold stresses. In order to study the role of FBA in photosynthesis and in regulating cold stress responses of tomato seedlings (Solanum lycopersicum ), we transformed a tomato inbred line (FF) with RNA interference (RNAi) vector containing SlFBA 7 reverse tandem repeat sequence. We found that the decreased SlFBA7 expression led to the decreased activities of FBA, as well as the activities of other main enzymes in the CBC. We also noticed a decrease in net photosynthetic rate, ribulose‐1,5‐bisphosphate and soluble sugar content, stem diameter, dry weight and seed size in RNAi SlFBA7 plants compared to wild‐type. However, there are no changes in starch contents in the RNAi transgenic plants. RNAi SlFBA7 plants showed a decreased germination rate, and an increased levels of superoxide anions (O₂^·‐) and hydrogen peroxide (H₂O₂) under low temperature (8/5°C) and low‐light intensity (100 μmol m^?2 s^?1 photon flux density) growth conditions. These findings demonstrated the important role of SlFBA7 in regulating growth and chilling tolerance of tomato seedlings, and suggested that the catalytic activity of FBA in the CBC is sensitive to temperature.     

Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance

The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens‐mediated transformation. Catalase activity in progeny from transgenic plants was approximately three‐fold higher than that in wild‐type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µm paraquat under moderate light intensity, whereas leaf discs from wild‐type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild‐type leaf discs at 24 h after treatment with 1 µm paraquat and 10 mm H₂O₂, respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wild‐type plants were sprayed with 100 µm paraquat and exposed to high light illumination (800 µmol m^?2 s^?1). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild‐type plants. The catalase activity and the photosynthesis activity (indicated by the F_v/F_m ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO₂ fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 °C) under high light intensity (1000 µmol m^?2 s^?1). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photo‐oxidative stress invoked by paraquat treatment, drought stress and chilling stress.     

Animal type 1 cryptochromes. Analysis of the redox state of the flavin cofactor by site-directed mutagenesis

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD(*-), in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD(ox) when purified, and the flavin is readily reduced to FAD(*-) by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD(*-) in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD(*-) represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

Nicotianamine synthase: Gene isolation,gene transfer and application for the manipulation of plant iron assimilation

Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilise iron, plants have evolved at least two different strategies. The non-proteinogenic amino acid nicotianamine is an essential component of both pathways.We briefly review the characterisation of the nicotianamine synthase as a member of a novel class of enzymes, the cloning of the corresponding gene coding sequences of barley, Arabidopsis and tomato as well as the molecular basis of the chloronerva mutant exhibiting severe defects in the regulation of iron metabolism.Further, we report on current experiments aiming to the application of various NAS-genes to manipulate iron assimilation in model and crop plants using transgenic sense and antisense approaches.