Time‐lapse imaging of Arabidopsis leaf development shows dynamic patterns of procambium formation

Abstract

Summary• The principles underlying the formation of leaf veins have long intrigued developmental biologists. In leaves, networks of vascular precursor procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Understanding dynamics of procambium formation has been hampered by the difficulty of observing the process in vivo .• Here we present a live-imaging technique that allows visual access to complex events occurring in developing leaves. We combined this method with stage-specific fluorescent markers in Arabidopsis ( Arabidopsis thaliana ) to visualize preprocambial strand formation and procambium differentiation during the undisturbed course of development and upon defined perturbations of vein ontogeny.• Under all experimental conditions, we observed extension, termination and fusion of preprocambial strands and simultaneous initiation of procambium differentiation along entire individual veins.• Our findings strongly suggest that progressiveness of preprocambial strand formation and simultaneity of procambium differentiation represent inherent properties of the mechanism underlying vein formation.

Expression of DOF genes identifies early stages of vascular development in Arabidopsis leaves

“…Under both undisturbed and pharmacologically perturbed leaf development, broad domains of DOF gene expression become restricted to sites of vein formation, suggesting that dynamics of DOF expression define reproducible cell states in preprocambial development. Our results assist in the mo-DOF5.3 pro :mGFP4er in 4-DAG first leaves as their venation is predominantly preprocambial and procambial (Donner et al, 2009;Sawchuk et al, 2007). Expression of fusions of DOF2.1, DOF4.6 and DOF5.3 was activated in correlation to zones of vein emergence, while DOF3.1 pro :HTA6:EYFP signals were restricted to the leaf tip and the most basal portion of the central midvein (F i g .…”

“…Initiation of ATHB8 expression is directly controlled by MP (Donner et al, 2009), and identifies acquisition of a critical and typically irreversible 'preprocambial' cell state that accurately predicts sites of vascular differentiation (Alonso-Peral et al, 2006;Candela et al, 2001;Carland and Nelson, 2004;Cnops et al, 2006;Donner et al, 2009;Kang and Dengler, 2004;Koizumi et al, 2000;Petricka and Nelson, 2007;Pineau et al, 2005;Sawchuk et al, 2007Sawchuk et al, , 2008Scarpella et al, 2004Scarpella et al, , 2006. A view of the transcriptional landscape of cells at preprocambial stages would be particularly valuable as it might provide insight into the molecular pathways underlying vein formation.…”

“…While onset of PIN1 and MP expression marks an unstable and reversible state in formation of leaf vascular strands (or 'veins'), lines of PIN1 and MP-expressing ground cells that are stabilized towards procambial fate activate expression of the class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) gene ATHB8 (Baima et al, 1995;Donner et al, 2009;Kang and Dengler, 2004;Sawchuk et al, 2007;Scarpella et al, 2004). Initiation of ATHB8 expression is directly controlled by MP (Donner et al, 2009), and identifies acquisition of a critical and typically irreversible 'preprocambial' cell state that accurately predicts sites of vascular differentiation (Alonso-Peral et al, 2006;Candela et al, 2001;Carland and Nelson, 2004;Cnops et al, 2006;Donner et al, 2009;Kang and Dengler, 2004;Koizumi et al, 2000;Petricka and Nelson, 2007;Pineau et al, 2005;Sawchuk et al, 2007Sawchuk et al, , 2008Scarpella et al, 2004Scarpella et al, , 2006.…”

Unique and Overlapping Expression Patterns among Members of Photosynthesis-Associated Nuclear Gene Families in Arabidopsis

“…Furthermore, because YFP signals can be easily separated from both CFP and GFP fluorescence in plants (e.g. Kato et al, 2002;Sawchuk et al, 2007), YFP fusions should greatly expand the number of possible combinations with preexisting fluorescent marker lines and consequently vastly increase the utility of the resources generated here. Targeting all the YFP produced in each individual cell to the nucleus locally increases the concentration of YFP of 30% to 50%, resulting in enhanced sensitivity of signal detection (Joly, 2007).…”

“…The origins of the Athb8 pro :ECFP:3xNLS and the Athb8 pro :HTA6:EYFP lines have been previously described (Sawchuk et al, 2007). To generate the RbcS pro :HTA6:EYFP and Lhc pro :HTA6:EYFP lines, sequences upstream of RbcS or Lhc coding regions, respectively, were amplified from Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) genomic DNA using Finnzymes Phusion high-fidelity DNA polymerase (New England BioLabs) and genespecific primers (Supplemental Table S1), integrated into pDONR221(Invitrogen) with BP clonase II (Invitrogen), sequence-checked, and recombined into the Gateway-adapted pFYTAG binary vector (Zhang et al, 2005) using LR clonase II (Invitrogen).…”