An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Siliplant1 protein precipitates silica in sorghum silica cells.

Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume using an unknown biological factor. Using bioinformatics tools, we identified a previously uncharacterized protein in Sorghum bicolor, which we named Siliplant1 (Slp1). Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. We found Slp1 RNA in sorghum immature leaf and immature inflorescence. In leaves, transcription was highest just before the active silicification zone (ASZ). There, Slp1 was localized specifically to developing silica cells, packed inside vesicles and scattered throughout the cytoplasm or near the cell boundary. These vesicles fused with the membrane, releasing their content in the apoplastic space. A short peptide that is repeated five times in Slp1 precipitated silica in vitro at a biologically relevant silicic acid concentration. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types. Our results show that Slp1 precipitates silica in sorghum silica cells.

Characterization of phloem-sap transcription profile in melon plants.

The phloem's role as a tissue responsible for the distribution of photoassimilates and nutrients among the various organs of higher plants has long been recognized. Recent studies have established that numerous proteins and mRNA molecules are also present in the phloem translocation stream; however, limited information is available on the identity of transcripts present within the phloem sap. In this study, a genomic approach was taken to produce a transcription profile of melon phloem sap. A cDNA library was constructed from mRNAs extracted from melon phloem sap and 1900 clones were randomly selected for sequencing. Selection of high-quality sequences resulted in 986 unique transcripts corresponding to 1830 ESTs. A comparison between the phloem-sap library and publicly available libraries from leaves and fruits indicated that the transcript profile of phloem sap is unique, with a substantially higher proportion of genes associated with biotic stimulus, response to stress, and metal-ion binding. Manual functional analyses revealed that over 40% of the transcripts are related to stress and defence responses, while over 15% of them are related to signal transduction. Out of the 1830 ESTs, only three were characterized as coding for chlorophyll-binding protein or ribulose bisphosphate carboxylase. Heterografting experiments established that six out of 43 examined transcripts are capable of long-distance trafficking from melon stocks to pumpkin scions. Annotation of these six transcripts revealed that three of them are associated with auxin-signal transduction while the other three were not identified. The potential role of the expressed transcripts in the phloem sap is discussed.

From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes.

Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species.

Long-distance trafficking of macromolecules in the phloem.

The fact that macromolecules such as proteins and mRNAs overcome the symplastic barriers between various tissue domains was first evidenced by the movement of plant viruses. We have recently demonstrated that viral infection disengages the symplastic restriction present between the sieve element-companion cell complex and neighboring cells in tobacco plants. As a result, green fluorescent protein, which was produced in mesophyll and bundle sheath cells, could traffic into the sieve tube and travel long distances within the vascular system. In this addendum we discuss the likely existence of a novel plant communication network in which macromolecules also act as long-distance trafficking signals. Plasmodesmata interconnecting sieve elements and companion cells as well as plasmodesmata connecting the sieve tube with neighboring cells may play a central role in establishing this communication network.