BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor‐like cytoplasmic kinase sub‐family (RLCK‐XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T‐DNA insertion lines for eight BSK genes (BSK1–BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24‐epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR‐responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3‐like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3‐like kinases.     

DWARF4 accumulation in root tips is enhanced via blue light perception by cryptochromes

Brassinosteroid (BR) signalling is known to be coordinated with light signalling in above ground tissue. Many studies focusing on the shade avoidance response in above ground tissue or hypocotyl elongation in darkness have revealed the contribution of the BR signalling pathway to these processes. We previously analysed the expression of DWARF 4 (DWF4), a key BR biosynthesis enzyme, and revealed that light perception in above ground tissues triggered DWF4 accumulation in root tips. To determine the required wavelength of light and photoreceptors responsible for this regulation, we studied DWF4‐GUS marker plants grown in several monochromatic light conditions. We revealed that monochromatic blue LED light could induce DWF4 accumulation in primary root tips and root growth as much as white light, whereas monochromatic red LED could not. Consistent with this, a cryptochrome1/2 double mutant showed retarded root growth under white light whereas a phytochromeA/B double mutant did not. Taken together, our data strongly indicated that blue light signalling was important for DWF4 accumulation in root tips and root growth. Furthermore, DWF4 accumulation patterns in primary root tips were not altered by auxin or sugar treatment. Therefore, we hypothesize that blue light signalling from the shoot tissue is different from auxin and sugar signalling.     

Brassinosteroid signaling modulates submergence-induced hyponastic growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The role of brassinosteroids (BRs) in hyponastic growth induced by submergence was investigated in Arabidopsis thaliana. Under flooding conditions, exogenously applied BRs increased hyponastic growth of rosette leaves. This hyponastic growth was reduced in a BR insensitive mutant (bri1-5), while it was increased in a BR dominant mutant (bes1-D). Further, expression of hypoxia marker genes, HRE1 and HRE2, was elevated in submerged bes1-D. These results indicate that BRs exert a positive action on hyponastic growth of submerged Arabidopsis leaves. Expression of ethylene biosynthetic genes, such as ACS6, ACS8 and ACO1, which are up-regulated by submergence, was also activated by application of BRs and in bes1-D. The enhanced hyponastic growth in submerged bes1-D was significantly reduced by application of cobalt ion, suggesting that BRs control hyponastic growth via ethylene, which seems to be synthesized by ACO6 and ACO8 followed by ACO1 in submerged leaves. A double mutant, bes1-Dxaco1-1, showed hyponastic growth activity similar to that seen in aco1-1, demonstrating that the BR signaling for regulation of hyponastic growth seems to be an upstream event in ethylene-induced hyponastic growth under submergence in Arabidopsis.     

Brassinosteroids are involved in ethylene‐induced Pst DC3000 resistance in Nicotiana benthamiana

• Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear.
• We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana.
• Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET‐induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ‐treated and gene‐silenced were completely eliminate this response.
• Our results suggest BR is involved in ET‐induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR‐dependent manner.

     

The effects of DELLAs on growth change with developmental stage and brassinosteroid levels

There are two stages in photomorphogenesis. First, seedlings detect light and open their cotyledons. Second, seedlings optimize their light environment by controlled elongation of the seedling stem or hypocotyl. In this study, we used time‐lapse imaging to investigate the relationship between the brassinosteroid (BR) and gibberellin (GA) hormones across both stages of photomorphogenesis. During the transition between one stage and the other, growth promotion by BRs and GAs switched from an additive to a synergistic relationship. Molecular genetic analysis revealed unexpected roles for known participants in the GA pathway during this period. Members of the DELLA family could either repress or enhance BR growth responses, depending on developmental stage. At the transition point for seedling growth dynamics, the BR and GA pathways had opposite effects on DELLA protein levels. In contrast to GA‐induced DELLA degradation, BR treatments increased the levels of REPRESSOR of ga1‐3 (RGA) and mimicked the molecular effects of stabilizing DELLAs. In addition, DELLAs showed complex regulation of genes involved in BR biosynthesis, implicating them in BR homeostasis. Growth promotion by GA alone depended on the PHYTOCHROME INTERACTING FACTOR (PIF) family of master growth regulators. The effects of BR, including the synergistic effects with GA, were largely independent of PIFs. These results point to a multi‐level, dynamic relationship between the BR and GA pathways.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> constitutive photomorphogenic mutant, <Emphasis Type="Italic">bls1</Emphasis>,displays altered brassinosteroid response and sugar sensitivity

We have isolated an Arabidopsis mutant impaired in light- and brassinosteroid (BR) induced responses, as well as in sugar signalling. The bls1 (brassinosteroid, light and sugar1) mutant displays short hypocotyl, expanded cotyledons, and de-repression of light-regulated genes in young seedlings, and leaf differentiation and silique formation on prolonged growth in dark. In light, the bls1 mutant is dwarf and develops a short root, compact rosette, with reduced trichome number, and exhibits delayed bolting. The activity of the BR inducible TCH4 and auxin inducible SAUR promoters, fused with GUS gene, is also altered in seedlings harbouring bls1 mutant background. In addition, the bls1 mutant is hypersensitive to metabolizable sugars. The short hypocotyl phenotype in dark, short root phenotype in light and sugar hypersensitivity could be rescued with BR application. Moreover, the bls1 mutant also showed higher expression of a BR biosynthetic pathway gene CPD, which is known to be feedback-regulated by BR. Using a genome-wide AFLP mapping strategy, the bls1 mutant has been mapped to a 1.4Mb region of chromosome 5. Since no other mutant with essentially a similar phenotype has been assigned to this region, we suggest that the bls1 mutant defines a novel locus involved in regulating endogenous BR levels, with possible ramifications in integrating light, hormone and sugar signalling.     

Photomorphogenesis of the root system in developing sunflower seedlings: a role for sucrose

• The domestic sunflower (Helianthus annuus L. cv. ‘Giganteus’) has been used since the 19th century as a model plant for the study of seedling development in darkness and white light (WL) (scoto‐ versus photomorphogenesis). However, most pertinent studies have focused on the developmental patterns of the hypocotyl and cotyledons, whereas the root system has been largely ignored.
• In this study, we analysed entire sunflower seedlings (root and shoot) and quantified organ development in the above‐ and belowground parts of the organism under natural (non‐sterile) conditions.
• We document that seedlings, raised in moist vermiculite, are covered with methylobacteria, microbes that are known to promote root development in Arabidopsis. Quantitative data revealed that during photomorphogenesis in WL, the root system expands by 90%, whereas stem elongation is inhibited, and hook opening/cotyledon expansion occurs. Root morphogenesis may be mediated via imported sucrose provided by the green, photosynthetically active cotyledons. This hypothesis is supported by the documented effect of sucrose on the induction of lateral root initials in sunflower cuttings. Under these experimental conditions, phytohormones (auxin, cytokinin, brassinolide) exerted little effect on root and cotyledon expansion, and no hormone‐induced initiation of lateral roots was observed.
• It is concluded that sucrose not only acts as an energy source to fuel cell metabolism but is also a shoot‐derived signalling molecule that triggers root morphogenesis.