Biology subjects

Vlad, D.

Publications and source records attributed to Vlad, D..

3 recordsLinked to original sources

bioRxiv2023

THE WIP6 TRANSCRIPTION FACTOR TOO MANY LATERALS SPECIFIES VEIN TYPE IN C4 AND C3 GRASS LEAVES

Grass leaves are invariantly strap shaped with an elongated distal blade and a proximal sheath that wraps around the stem. Underpinning this uniform shape is a scaffold of leaf veins, most of which extend in parallel lines along the proximo-distal leaf axis. Differences between species are apparent both in the types of veins that develop and in the spacing between them across the medio-lateral leaf axis. A prominent engineering goal is to increase vein density and the proportion of bundle sheath cells surrounding the veins in leaves of C3 photosynthesizing species such as rice, in order to facilitate introduction of the more efficient C4 photosynthetic pathway. Here we discover that the WIP6 zinc finger transcription factor TOO MANY LATERALS (TML) specifies vein rank in both maize (C4) and rice (C3), species with distinct venation patterns. Loss of function tml mutations lead to the development of large lateral veins in positions normally occupied by smaller intermediate veins. The spatial localization of TML transcripts in wild-type leaves is consistent with a role in suppressing lateral vein formation in procambial cells that develop intermediate veins, specifically the class of intermediate veins that extend from the leaf blade into the leaf sheath. Attempts to manipulate TML function in rice were unsuccessful because transgene expression was silenced, suggesting that precise spatial and temporal regulation of TML expression is essential during the regeneration of shoot tissue from callus. Given that transcriptome analysis demonstrated altered profiles of genes associated with cytokinin and auxin signaling in loss of function maize mutants, the necessity for tight regulation of TML gene expression could be an indirect consequence of hormonal inbalances as opposed to ectopic activity of a specific downstream target. Importantly, however, loss of function mutants in rice display increased vascular and bundle sheath cell occupancy in the leaf. Collectively this work provides an understanding of how vein rank is specified in grass leaves and a first step towards an anatomical chassis for C4 engineering in rice.

plant biology↗

bioRxiv2022

DEFECTIVELY ORGANIZED TRIBUTARIES 5 is not required for leaf venation patterning in Arabidopsis thaliana

The search for genetic regulators of leaf venation patterning started over thirty years ago, primarily focussed on mutant screens in the eudicotyledon Arabidopsis thaliana. Developmental perturbations in either cotyledons or true leaves led to the identification of transcription factors required to elaborate the characteristic reticulated vein network. An ortholog of one of these, the C2H2 Zn finger protein DEFECTIVELY ORGANIZED TRIBUTARIES 5 (AtDOT5), was recently identified through transcriptomics as a candidate regulator of parallel venation in maize leaves. To elucidate how AtDOT5 orthologs regulate vein patterning, we generated three independent loss of function mutations by gene editing in Arabidopsis. Surprisingly, none of them exhibited any obvious phenotypic perturbations. To reconcile our findings with earlier reports, we re-evaluated the original Atdot5-1 and Atdot5-2 alleles. By genome sequencing, we show that reported mutations at the Atdot5-1 locus are actually polymorphisms between Landsberg erecta and Columbia ecotypes, and that other mutations present in the background most likely cause the pleiotropic mutant phenotype observed. We further show that a T-DNA insertion in the Atdot5-2 locus has no impact on leaf venation patterns when segregated from other T-DNA insertions present in the original line. We thus conclude that AtDOT5 plays no role in leaf venation patterning in Arabidopsis. Significance statementAn understanding of gene function is often derived on the basis of loss of function mutant phenotypes and thus correct identification of mutated loci is crucial. Through gene editing we reveal previous mis-identification of a causative mutation that led to inappropriate functional assignation for the DEFECTIVELY ORGANIZED TRIBUTARIES 5 gene in Arabidopsis thaliana.

plant biology↗

bioRxiv2019

Optimization of a Modular Steroid-Inducible Gene Expression System for Use in Rice

Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology. Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme {beta}-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with six hours of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01M DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl {beta}-D-1-thiogalactopyranoside (IPTG). The system is thus suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

plant biology↗