The Arabidopsis thaliana E3 Ubiquitin Ligase BRIZ Functions in Abscisic Acid Response

The ubiquitin system is essential for multiple hormone signaling pathways in plants. Here, we show that the Arabidopsis thaliana E3 ligase BRIZ, a heteromeric ligase that consists minimally of BRIZ1 and BRIZ2 proteins, functions in abscisic acid (ABA) signaling or response. briz1 and briz2 homozygous mutants either fail to germinate or emerge later than wild-type seedlings, with little cotyledon expansion or root elongation and no visible greening. Viability staining indicates that briz1 and briz2 embryos are alive but growth-arrested. Germination of briz mutants is improved by addition of the carotenoid biosynthetic inhibitor fluridone or gibberellic acid (GA3), and briz mutants have improved development in backgrounds deficient in ABA synthesis (gin1-3/aba2) or signaling (abi5-7). Endogenous ABA is not higher in briz2 seeds compared to wild-type seeds, and exogenous ABA does not affect BRIZ mRNAs in imbibed seeds. These results indicate that briz embryos are hypersensitive to ABA and that under normal growth conditions, BRIZ acts to suppress ABA signaling or response. ABA signaling and sugar signaling are linked, and we found that briz1 and briz2 mutants excised from seed coats are hypersensitive to sucrose. Although briz single mutants do not grow to maturity, we were able to generate mature briz2-3 abi5-7 double mutant plants that produced seeds. These seeds are more sensitive to exogenous sugar and are larger than seeds from sibling abi5-7 BRIZ2/briz2-3 plants, suggesting that BRIZ has a parental effect on seed development. From these data, we propose a model in which the BRIZ E3 ligase suppresses ABA responses during seed maturation and germination and early seedling establishment.

The CBL-Interacting Protein Kinase NtCIPK23 Positively Regulates Seed Germination and Early Seedling Development in Tobacco (Nicotiana tabacum L.)

CBL-interacting protein kinase (CIPK) family is a unique group of serine/threonine protein kinase family identified in plants. Among this family, AtCIPK23 and its homologs in some plants are taken as a notable group for their importance in ions transport and stress responses. However, there are limited reports on their roles in seedling growth and development, especially in Solanaceae plants. In this study, NtCIPK23, a homolog of AtCIPK23 was cloned from Nicotiana tabacum. Expression analysis showed that NtCIPK23 is mainly expressed in the radicle, hypocotyl, and cotyledons of young tobacco seedlings. The transcriptional level of NtCIPK23 changes rapidly and spatiotemporally during seed germination and early seedling growth. To study the biological function of NtCIPK23 at these stages, the overexpressing and CRISPR/Cas9-mediated knock-out (ntcipk23) tobacco lines were generated. Phenotype analysis indicated that knock-out of NtCIPK23 significantly delays seed germination and the appearance of green cotyledon of young tobacco seedling. Overexpression of NtCIPK23 promotes cotyledon expansion and hypocotyl elongation of young tobacco seedlings. The expression of NtCIPK23 in hypocotyl is strongly upregulated by darkness and inhibited under light, suggesting that a regulatory mechanism of light might underlie. Consistently, a more obvious difference in hypocotyl length among different tobacco materials was observed in the dark, compared to that under the light, indicating that the upregulation of NtCIPK23 contributes greatly to the hypocotyl elongation. Taken together, NtCIPK23 not only enhances tobacco seed germination, but also accelerate early seedling growth by promoting cotyledon greening rate, cotyledon expansion and hypocotyl elongation of young tobacco seedlings.

A Synthetic Peptide Encoded by a Random DNA Sequence Inhibits Discrete Red Light Responses

We have identified a synthetic peptide that interrupts discrete aspects of seedling development under red light. Previous reports have demonstrated that plants transformed with random DNA sequences produce synthetic peptides that affect plant biology. In this report one specific peptide is characterized that inhibits discrete aspects of red-light-mediated Arabidopsis thaliana development during photomorphogenesis. Seedlings expressing the PEP6-32 peptide presented longer hypocotyls and diminished cotyledon expansion when grown under red light. Other red-light-mediated seedling processes such as induction of Lhcb (cab) transcripts or loss of vertical growth remained unaffected. Long-term responses to red light in PEP6-32 expressing plants, such as repression of flowering time, did not show defects in red light signaling or integration. A synthesized peptide applied exogenously induced the long-hypocotyl phenotype under red light in non-transformed seedlings. The results indicate that the PEP6-32 peptide causes discrete cell expansion defects during early seedling development in red light, mimicking weak phyB alleles in some aspects of seedling photomorphogenesis. The findings demonstrate that new chemistries derived from random peptide expression can modulate specific facets of plant growth and development.One Sentence SummaryA plant line expressing random DNA sequence expresses a synthetic peptide that affects specific red-light responses in a developing seedling.

Arabidopsis SAURs are critical for differential light regulation of the development of various organs

During deetiolation of Arabidopsis seedlings, light promotes the expansion of cotyledons but inhibits the elongation of hypocotyls. The mechanism of this differential regulation of cell enlargement is unclear. Our organ-specific transcriptomic analysis identified 32 Small Auxin Up RNA (SAUR) genes whose transcripts were light-induced in cotyledons and/or repressed in hypocotyls. We therefore named these SAURs as lirSAURs. Both overexpression and mutation analyses demonstrated that lirSAURs could promote cotyledon expansion and opening and enhance hypocotyl elongation, possibly by inhibiting phosphatase activity of D-clade type 2C protein phosphatases (PP2C-Ds). Light reduced auxin levels to down-regulate the expression of lirSAURs in hypocotyls. Further, phytochrome-interacting factors (PIFs) were shown to directly bind the genes encoding these SAURs and differentially regulate their expression in cotyledons and hypocotyls. Together, our study demonstrates that light mediates auxin levels and PIF stability to differentially regulate the expression of lirSAURs in cotyledons and hypocotyls, and these lirSAURs further mediate the differential growth of these two organs.

Constitutive expression of the barley dehydrin gene aba2 enhances Arabidopsis germination in response to salt stress

Dehydrins (DHNs) are a sub-family of the late embryogenesis abundant proteins generally induced during development of desiccation tolerance in seeds and water deficit or salinity stress in plants. Nevertheless, a detailed understanding of the DHNs function is still lacking. In this work we investigated the possible protective role during salt stress of a <em>Dhn</em> from <em>Hordeum vulgare</em> (L.), <em>aba2</em>. The coding sequence of the <em>aba2</em> gene was constitutively expressed in transgenic lines of <em>Arabidopsis thaliana</em> (L.). During salt stress conditions germination rate, cotyledon expansion and greening were greatly improved in the transgenic lines as compared to the wild type. Between 98 and 100% of the transgenic seeds germinated after two weeks in media containing up to 250 mM NaCl, and 90% after 22 days at 300 mM NaCl. In conditions of 200 mM NaCl 93% of the transgenic cotyledons had greened after two weeks, outperforming the wild type by 45%. Our study provides further evidence that DHNs have an important role in salt stress tolerance. The production of plants constitutively expressing DHNs could be an effective strategy to improve plant breeding programs.

Bioreactors in coffee micropropagation

In coffee, bioreactors are the most promising way for scaling-up micropropagation processes, particularly somatic embryogenesis. The availability of an efficient somatic embryogenesis process would allow the rapid mass production of heterozygous materials such as selected Coffea canephora clones and F1 Arabica hybrid varieties. For the last fifteen years, bioreactors (mechanically or pneumatically agitated bioreactors, temporary immersion bioreactors) have mostly been used on coffee to optimize the mass regeneration of somatic embryos from embryogenic tissues. This review presents the main results, obtained with several bioreactor models, concerning the different steps of the micropropagation process : i) the multiplication of embryogenic tissues, ii) the somatic embryo mass regeneration and iii) the production of pre-germinated embryos and plantlets in bioreactors. The literature shows that scaling-up can be successful, since very efficient embryo production has been achieved for both C. arabica and C. canephora. Moreover, it was proven that the pre-germinated coffee embryos - i.e. embryonic axis elongation (10-12 mm), root tip formation, cotyledon expansion and greening - obtained in temporary immersion bioreactors were photoautotrophic and able to regenerate vigorous plantlets after sowing under nursery conditions. The feasibility to apply the bioreactor technology in an industrial micropropagation procedure is also discussed in the particular socio-economic context of coffee growing.

Synthesis and preliminary bioactivity evaluation of new pregnane brassinosteroid-like compounds

Seven new pregnane compounds bearing some representative A- and B-ring brassinosteroid functions, as well as hydrogen bond donor and acceptor ones on the D ring, were efficiently synthesized. The obtained compounds did not show remarkable plant growth-promoting activity in the radish hypocotyl elongation and cotyledon expansion bioassays, however, introducing oxygen and amino functions on the D ring led to an enhancement of the bioactivity. The 16β-functionalized pregnane brassinosteroid-like compounds were slightly more active than the 16α-functionalized ones.Key words: steroids, brassinosteroids, pregnane analogues.