Comparative studies on the responses to salt stress of taxonomically related taxa should help to elucidate relevant mechanisms of stress tolerance in plants. We have applied this strategy to three Plantago species adapted to different natural habitats, P. crassifolia and P. coronopus–both halophytes–and P. major, considered as salt-sensitive since it is never found in natural saline habitats. Growth inhibition measurements in controlled salt treatments indicated, however, that P. major is quite resistant to salt stress, although less than its halophytic congeners. The contents of monovalent ions and specific osmolytes were determined in plant leaves after four-week salt treatments. Salt-treated plants of the three taxa accumulated Na+ and Cl- in response to increasing external NaCl concentrations, to a lesser extent in P. major than in the halophytes; the latter species also showed higher ion contents in the non-stressed plants. In the halophytes, K+ concentration decreased at moderate salinity levels, to increase again under high salt conditions, whereas in P. major K+ contents were reduced only above 400 mM NaCl. Sorbitol contents augmented in all plants, roughly in parallel with increasing salinity, but the relative increments and the absolute values reached did not differ much in the three taxa. On the contrary, a strong (relative) accumulation of proline in response to high salt concentrations (600–800 mM NaCl) was observed in the halophytes, but not in P. major. These results indicate that the responses to salt stress triggered specifically in the halophytes, and therefore the most relevant for tolerance in the genus Plantago are: a higher efficiency in the transport of toxic ions to the leaves, the capacity to use inorganic ions as osmotica, even under low salinity conditions, and the activation, in response to very high salt concentrations, of proline accumulation and K+ transport to the leaves of the plants.

The COP9 signalosome (CSN) is an evolutionary conserved multiprotein complex that regulates many aspects of plant development by controlling the activity of CULLIN-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate and target for proteasomal degradation a vast number of specific substrate proteins involved in many developmental and physiological processes, including light and hormone signaling and cell division. As a consequence of CSN pleiotropic function, complete loss of CSN activity results in seedling lethality. Therefore, a detailed analysis of CSN physiological functions in adult Arabidopsis plants has been hampered by the early seedling lethality of csn null mutants. Here we report the identification and characterization of a viable allele of the Arabidopsis COP9 signalosome subunit 4 (CSN4). The allele, designated csn4-2035, suppresses the adventitious root (AR) phenotype of the Arabidopsis superroot2-1 mutant, potentially by altering its auxin signaling. Furthermore, we show that although the csn4-2035 mutation affects primary and lateral root (LR) formation in the 2035 suppressor mutant, CSN4 and other subunits of the COP9 complex seem to differentially control AR and LR development.

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Salt stress responses were studied in three Plantago species (P. crassifolia, P. coronopus and P. major), with different levels of salt stress tolerance, at growth and reproductive stages of the life cycle. Plants were treated with increasing salt concentrations (0, 100, 200 and 400 mM NaCl) under controlled conditions in the greenhouse. Fresh biomass, leaf length and reproductive parameters related to flowering were analysed. To evaluate the reproductive success of salt-stressed plants, seeds produced after two months of treatment were sampled, and the mean number of seeds per plant and mean seed weight per treatment were determined. Seeds were germinated in vitro and seed and seedling quality were assessed by determining germination percentages and rates, length of radicle, hypocotyl, cotyledon leaves, and the angle of cotyledonary leaves. The 'seedling vigour index' was also calculated. Vegetative (fresh weight and leaf length) and reproductive traits (number of inflorescences, scape and spike length, mean number of seeds per spike and mean seed weight) were less affected by salinity in the more stress tolerant species P. crassifolia and P. coronopus than in P. major. Rate and velocity of germination of seeds produced by plants grown under saline treatments were similar in all species; however seedling development was less successful in P. major, as shown by length of the radicle, hypocotyl and cotyledonary leaves. The relative sensitivity to salt of the investigated Plantago species correlated with their distribution in nature: P. major, never present in saline environments, was much more affected by salt than its halophytic counterparts, while P. crassifolia, which grows in saline habitats, exhibited the most efficient responses to salt stress, especially at the early seedling development stage.

Map-based cloning (MBC) is the conventional approach for linking phenotypes to genotypes, and has been successfully used to identify causal mutations in diverse organisms. Next-generation sequencing (NGS) technologies offer unprecedented possibilities to sequence the entire genomes of organisms, thereby in principle enabling direct identification of causal mutations without mapping. However, although mapping-by-sequencing has proven to be a cost effective alternative to classical MBC in particular situations, methods based solely on NGS still have limitations and need to be refined. Aiming to identify the causal mutations in suppressors of Arabidopsis thaliana superroot2 phenotype, generated by ethyl methane sulfonate (EMS) treatment, we combined NGS and classical mapping, to rapidly identify the point mutations and restrict the number of testable candidates by defining the chromosomal intervals containing the causal mutations, respectively. The NGS-assisted mapping approach we describe here facilitates unbiased identification of virtually any causal EMS-generated mutation by overlapping the identification (deep sequencing) and validation (mapping) steps. To exemplify the useful marriage of the two approaches we discuss the strategy used to identify a new viable recessive allele of the Arabidopsis CULLIN1 gene in the non-reference Wassilewskija (Ws-4) accession.

In the original version of this Article, some instances of "CSN" were incorrectly given as "CNS". This error has now been corrected in the PDF and HTML versions of the Article.

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