With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt-tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty-six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt-secretion capacity in crops.

Limonium bicolor, a typical recretohalophyte that lives in saline environments, excretes excessive salt to the environment through epidermal salt glands to avoid salt stress. The aim of this study was to screen for L. bicolor genes involved in salt secretion by high-throughput RNA sequencing. We established the experimental procedure of salt secretion using detached mature leaves, in which the optimal salt concentration was determined as 200 mM NaCl. The detached salt secretion system combined with Illumina deep sequencing were applied. In total, 27,311 genes were annotated using an L. bicolor database, and 2040 of these genes were differentially expressed, of which 744 were up-regulated and 1260 were down-regulated with the NaCl versus the control treatment. A gene ontology enrichment analysis indicated that genes related to ion transport, vesicles, reactive oxygen species scavenging, the abscisic acid-dependent signaling pathway and transcription factors were found to be highly expressed under NaCl treatment. We found that 102 of these genes were likely to be involved in salt secretion, which was confirmed using salt-secretion mutants. The present study identifies the candidate genes in the L. bicolor salt gland that are highly associated with salt secretion. In addition, a salt-transporting pathway is presented to explain how Na(+) is excreted by the salt gland in L. bicolor. These findings will shed light on the molecular mechanism of salt secretion from the salt glands of plants.

BackgroundThe genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the genus Flaveria contains 21 of the 23 known Flaveria species and has been previously constructed using a combination of morphological data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnL-F).ResultsHere we developed a new strategy to update the phylogenetic tree of 16 Flaveria species based on RNA-Seq data. The updated phylogeny is largely congruent with the previously published tree but with some modifications. We propose that the data collection method provided in this study can be used as a generic method for phylogenetic tree reconstruction if the target species has no genomic information. We also showed that a “F. pringlei” genotype recently used in a number of labs may be a hybrid between F. pringlei (C3) and F. angustifolia (C3-C4).ConclusionsWe propose that the new strategy of obtaining phylogenetic sequences outlined in this study can be used to construct robust trees in a larger number of taxa. The updated Flaveria phylogenetic tree also supports a hypothesis of stepwise and parallel evolution of C4 photosynthesis in the Flavaria clade.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0399-9) contains supplementary material, which is available to authorized users.

C 4 photosynthesis is a complex trait that evolved from its ancestral C 3 photosynthesis by recruiting pre-existing genes. These co-opted genes were changed in many aspects compared to their counterparts in C 3 species. Most of the evolutionary changes of the C 4 shuttle enzymes are well characterized, however, evolutionary changes for the recruited metabolite transporters are less studied. Here we analyzed the evolutionary changes of the shuttle enzyme phosphoenolpyruvate (PEP) transporter (PPT) during its recruitment from C 3 to C 4 photosynthesis. Our analysis showed that among the two PPT paralogs PPT1 and PPT2, PPT1 was the copy recruited for C 4 photosynthesis in multiple C 4 lineages. During C 4 evolution, PPT1 gained increased transcript abundance, shifted its expression from predominantly in root to in leaf and from bundle sheath cell to mesophyll cell, and gained more rapid and long-lasting responsiveness to light. Modifications occurred in both regulatory and coding regions in C 4 PPT1 as compared to C 3 PPT1, however, the PEP transporting function of PPT1 remained. We found that PPT1 of a Flaveria C 4 species recruited a MEM1 B submodule in the promoter region, which might be related to the increased transcript abundance of PPT1 in C 4 mesophyll cells. The case study of PPT further suggested that high transcript abundance in a proper location is of high priority for PPT to support C 4 function.