Identification of MeC3HDZ1/MeCNA as a potential regulator of cassava storage root development.

The storage root (SR) of cassava is the main staple food in sub-Saharan Africa, where it feeds over 500 million people. However, little is known about the genetic and molecular regulation underlying its development. Unraveling such regulation would pave the way for biotechnology approaches aimed at enhancing cassava productivity. Anatomical studies indicate that SR development relies on the massive accumulation of xylem parenchyma, a cell-type derived from the vascular cambium. The C3HDZ family of transcription factors regulate cambial cells proliferation and xylem differentiation in Arabidopsis and other species. We thus aimed at identifying C3HDZ proteins in cassava and determining whether any of them shows preferential activity in the SR cambium and/or xylem. Using phylogeny and synteny studies, we identified eight C3HDZ proteins in cassava, namely MeCH3DZ1-8. We observed that MeC3HDZ1 is the MeC3HDZ gene displaying the highest expression in SR and that, within that organ, the gene also shows high expression in cambium and xylem. In-silico analyses revealed the existence of a number of potential C3HDZ targets displaying significant preferential expression in the SR. Subsequent Y1H analyses proved that MeC3HDZ1 can bind canonical C3HDZ binding sites, present in the promoters of these targets. Transactivation assays demonstrated that MeC3HDZ1 can regulate the expression of genes downstream of promoters harboring such binding sites, thereby demonstrating that MeC3HDZ1 has C3HDZ transcription factor activity. We conclude that MeC3HDZ1 may be a key factor for the regulation of storage root development in cassava, holding thus great promise for future biotechnology applications.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium.

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.

Extremophilic bacteria restrict the growth of Macrophomina phaseolina by combined secretion of polyamines and lytic enzymes.

Extremophilic microorganisms were screened as biocontrol agents against two strains of Macrophomina phaseolina (Mp02 and 06). Stenotrophomonas sp. AG3 and Exiguobacterium sp. S58 exhibited a potential in vitro antifungal effect on Mp02 growth, corresponding to 52.2% and 40.7% inhibition, respectively. This effect was confirmed by scanning electron microscopy, where images revealed marked morphological alterations in fungus hyphae. The bacteria were found to secrete lytic enzymes and polyamines. Exiguobacterium sp. S56a was the only strain able to reduce the growth of the two strains of M. phaseolina through their supernatant. Antifungal supernatant activity was correlated with the ability of bacteria to synthesize and excrete putrescine, and the exogenous application of this polyamine to the medium phenocopied the bacterial antifungal effects. We propose that the combined secretion of putrescine, spermidine, and lytic enzymes by extremophilic microorganism predispose these microorganisms to reduce the disease severity occasioned by M. phaseolina in soybean seedlings.

Conservation of Thermospermine Synthase Activity in Vascular and Non-vascular Plants.

In plants, the only confirmed function for thermospermine is regulating xylem cells maturation. However, genes putatively encoding thermospermine synthases have been identified in the genomes of both vascular and non-vascular plants. Here, we verify the activity of the thermospermine synthase genes and the presence of thermospermine in vascular and non-vascular land plants as well as in the aquatic plant Chlamydomonas reinhardtii. In addition, we provide information about differential content of thermospermine in diverse organs at different developmental stages in some vascular species that suggest that, although the major role of thermospermine in vascular plants is likely to be xylem development, other potential roles in development and/or responses to stress conditions could be associated to such polyamine. In summary, our results in vascular and non-vascular species indicate that the capacity to synthesize thermospermine is conserved throughout the entire plant kingdom.

High levels of auxin signalling define the stem-cell organizer of the vascular cambium.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium1. Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown2. Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium3. Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.