Developmentally equivalent tissue sampling based on growth kinematic profiling of Arabidopsis inflorescence stems

Abstract

Summary• Directional growth in Arabidopsis thaliana during bolting of the inflorescence stem makes this an attractive system for study of the underlying processes of tissue elongation and cell wall extension. Analysis of local molecular events accompanying Arabidopsis inflorescence stem elongation is hampered by difficulties in isolating developmentally matched tissue samples from different plants.• Here, we present a novel sampling approach in which specific developmental stages along the developing stem are defined nonintrusively in terms of their relative elemental growth rate by use of time-lapse imagery and subsequent derivation of growth kinematic profiles for individual plants.• Growth kinematic profiling reveals that key developmental transitions such as the point of maximum elongation rate and the point of cessation of elongation occur over broad and overlapping ranges across individuals within a population of the Columbia (Col-0) ecotype. The position of these transitions is only weakly correlated with overall plant height, which undermines the common assumption that physically similar plants have closely matched growth profiles.• This kinematic profiling approach provides high-resolution growth phenotyping of the developing stem and thereby enables the harvest, pooling and analysis of developmentally matched tissue samples from multiple Arabidopsis plants.

Gradients in Wall Mechanics and Polysaccharides along Growing Inflorescence Stems

“…Our results show that the apical-to-basal decrease in elongation rate along the Arabidopsis inflorescence stem, measured here in relatively young inflorescences and in previous studies in older stems (Suh et al, 2005;Hall and Ellis, 2012), is accompanied by wall stiffening, quantified as decreasing elastic and plastic compliances in stress/strain measurements, and by reduced polysaccharide mobility, weaker water-polysaccharide interactions, less branching, and reduced esterification of pectins, as seen in the SSNMR data. However, as in the case of maize roots (Beusmans and Silk, 1988), the distribution of growth rate did not parallel the distribution of wall compliances along the inflorescence stem.…”

“…Recent work has pointed to the advantages of the Arabidopsis inflorescence stem, which is readily marked for growth analysis and provides a powerful system for studying cell development (Suh et al, 2005;Hall and Ellis, 2012). We find that the inflorescence stem also is amenable to mechanical tests, to analysis of wall structure by small-angle neutron scattering (SANS), and to 13 C enrichment, enabling highresolution, multidimensional magic-angle-spinning (MAS) solid-state NMR (SSNMR) spectroscopy to examine wall polysaccharide structures and dynamics along the growth zone, with minimal disruption of cell walls.…”

Transcriptional programming during cell wall maturation in the expanding Arabidopsis stem

“…In order to position transcript profiles accurately within the cell wall expansion continuum, we employed growth kinematic profiling to establish relative elemental growth rates (REGRs) for contiguous stem segments harvested from a series of individual plants [15]. These profiles allowed us to identify three developmental stages for each plant being sampled: 1) an apical region where tissues begin to differentiate, and directional cell growth is initializing (termed ‘young’, or YNG), 2) a region where directional growth of the stem is most rapid (termed 'maximum growth-rate’, or MGR), and 3) a region where elongative growth is finishing (termed 'cessation', or CSS).…”

“…A number of inflorescence stem profiling studies have attempted to compare the global transcriptional changes occurring between specific developmental stages [4,5,7,14], but the experimental strategies employed have typically compared tissues from visually selected regions of multiple plants, and have operated under two untested assumptions: 1) that the pooled plants whose stems are being sampled all have similar developmental proportioning, and 2) that the sampling guidelines for the harvested plants, derived from destructive analysis of a different set of plants, accurately associate features such as appearance of lignification in the interfascicular fibres of the stem [4] with specific developmental growth stages. Contrary to these assumptions, growth kinematic profiling of expanding inflorescence stems of individual Arabidopsis plants has recently demonstrated that stem growth profiles actually vary widely from plant to plant, even within genetically homogeneous populations [15]. As a consequence, data obtained from indirectly selected and harvested stem regions are likely to be relatively poorly correlated with onset of processes involved in cell wall extension or modification events associated with specific growth stages.…”

“…In this study, we have applied growth kinematic profiling (GKP) to a series of individual Arabidopsis inflorescence stems as described in Hall and Ellis (2012) [15], and used the resulting growth rate profiles to generate pooled samples of stem tissue that accurately represent four discrete growth stages along the cell wall expansion developmental continuum. Our use of GKP-based sampling was expected to reduce the biological noise associated with indirect sampling strategies used in previous studies, and thus increase the sensitivity (power to detect actual differential expression) and accuracy of transcript profiling.…”