RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 and the stress-induced morphogenetic response

Abstract

Plants exposed to abiotic stress show a range of morphogenetic responses, sometimes termed the stress-induced morphogenetic response (SIMR). SIMR is principally composed of three components: inhibition of cell elongation, alterations in cell differentiation, and stimulus of cell division in localized areas. An explanation proposed for SIMR has been increased accumulation of reactive oxygen species (ROS) and alterations in hormone signaling. Mutations in the Arabidopsis thaliana RADICAL-INDUCED CELL DEATH1 (RCD1) gene have altered abiotic stress responses and ROS accumulation. Even in the absence of exogenous stress, these plants show many morphological changes also seen in SIMR. In the September issue of Plant Physiology we reported an in depth analysis of the phenotype of rcd1-3 plants as well as the phenotype of a mutations in the previously uncharacterized paralog of RCD1, SIMILAR TO RCD ONE1 (SRO1). sro1-1 plants have mild morphological changes and abiotic stress response defects while rcd1-3; sro1-1 double mutant plants have severe developmental defects, including less cell elongation. In this Addendum, we hypothesize that rcd1, sro1 and rcd1; sro1 mutant plants are under constitutive stress, and that this stress is responsible for at least some of the developmental defects seen in these plants.

Plants as sessile organisms cannot move upon environmental change. Therefore, plants have evolved a diverse repertoire of responses in order to lower stress exposure, limit the damage caused or repair such damage. Chronic mild stress, from a variety of abiotic stresses, can cause a morphogenetic response that has been termed the stress-induced morphogenetic response (SIMR).¹ This response involves growth inhibition through suppression of cell elongation, changes in cell differentiation status, and localized stimulation of cell division. Typical SIMR responses include decreased elongation of the primary root accompanied by increased formation of lateral roots, decreased stem height, decreased leaf area and increased branching. Importantly, plants do not cease growth, rather they redistribute the areas undergoing active growth.

Although the molecular and cellular network underlying SIMR has not been completely worked out, several key elements have been identified.² The importance of the phytohormone auxin in morphogenetic changes seen in SIMR has been noted by several groups. Changes in auxin distribution and metabolism are induced by many stresses and correlates well with phenotypes induced by stress, such as increased lateral root growth, suggesting that changes in auxin signaling may be a causative agent in SIMR. Furthermore, reactive oxygen species (ROS) are known to accumulate in plants upon stress of many types, especially those that have been linked with SIMR. Once ROS accumulates, plants upregulate ROS scavenging systems, which can subsequently provide protection against a range of further environmental assaults. Extensive interactions between the auxin signaling pathway and ROS have been documented, suggesting that these two pathways may act in concert during SIMR.

Mutations in the Arabidopsis thaliana gene RADICAL-INDUCED CELL DEATH1 (RCD1) were originally isolated in a screen for plants hypersensitive to ozone.³ This gene encodes a putative poly(ADP-ribose) polymerase (PARP).⁴ PARPs attach ADP-ribose subunits from NAD⁺ to proteins post-translationally and are found across the eukaryotes. Although members of this enzyme family share the PARP catalytic domain, other regions of the proteins can vary dramatically, reflecting the diversity of functions these proteins have acquired. RCD1 belongs to a group of PARPs found only in land plants (Citarelli, Teotia S and Lamb RS, submitted) and contains a WWE domain N-terminal to the PARP catalytic domain. RCD1 has been shown to have complex roles in abiotic stress and development. rcd1 mutants are known to accumulate, even under non-inducing conditions, ROS³ and nitric oxide,⁵ suggesting that it normally works, directly or indirectly, to negatively regulate the accumulation of these compounds. Further evidence that rcd1 plants may be under stress include upregulation in the mutant of AOX1a and UPOX, two markers of oxidative stress.⁶ Complicating any interpretation of defects seen in rcd1 single mutants is the fact that, in addition to RCD1, Arabidopsis also encodes a paralog, SIMILAR TO RCD ONE1 (SRO1).

In our recent publication,⁷ we describe in detail phenotypes of mutations in RCD1 and SRO1 and double mutants between the two. The developmental defects seen in the single mutants are similar to those associated with SIMR, although defects in RCD1 generally cause more severe defects. Both rcd1-3 and sro1-1 plants have an increased number of lateral roots (increase in local cell division and redirected growth), while rcd1-3 plants also have shorter primary roots. rcd1-3 plants are shorter with smaller leaves (growth inhibition). Examination of double mutant plants further support the hypothesis that many phenotypes seen when these genes are malfunctioning are due to deregulated SIMR. Most rcd1-3; sro1-1 plants die during embryogenesis; however, those that survive have severe defects. These plants are extremely short, due, at least in part, to reduced cell elongation in the stem. The leaves are small for similar reasons. In addition, these plants are bushy due to arrest of the shoot apical meristem and activation of axillary meristems. All of these phenotypes are extreme examples of phenotypes seen in plants under stress from a variety of sources, including UV-B, heavy metals and salt.¹

In order to determine if rcd1-3; sro1-1 seedlings grown under normal conditions are under stress, we examined molecular markers of stress in this background. The small ubiquitin-like modifier (SUMO) is a ubiquitin-like polypeptide attached covalently to proteins. In Arabidopsis it has been demonstrated that sumoylated proteins accumulate under a variety of abiotic stresses such as heat shock and H₂O₂.⁸ We examined the accumulation of SUMO-modified proteins in rcd1-3; sro1-1 seedlings in comparison to wild type and two mutant backgrounds (nuclear pore anchor (nua)-1 and -2) in which such proteins have previously been shown to accumulate (Fig. 1A; western done according to⁹). The double mutant seedlings accumulate more sumoylated proteins, not only in comparison to wild type but also in comparison to the nua mutants. The accumulation of modified proteins supports the hypothesis that rcd1-3; sro1-1 seedlings are exhibiting constitutive stress. The expression of PARP2, which encodes a so-called classical PARP enzyme involved in DNA repair,¹⁰ has been shown to go up under a number of stress conditions.^11–15 We used RT-PCR to examine expression of this gene in our mutant backgrounds. PARP2 expression is increased in rcd1-3; sro1-1 seedlings and may also be higher than wild type in rcd1-3 and sro1-1 single mutants (Fig. 1B). This further supports our contention that loss of function in RCD1 and SRO1 results in constitutive stress and morphogenetic defects similar to those seen in SIMR.

Figure 1.

rcd1-3; sro1-1 plants are under constitutive stress. (A) rcd1-3; sro1-1 seedlings accumulate sumoylated proteins. The upper panel shows a western blot with anti-SUM O antibody according to Xu et al.⁹ Asterisk indicates sumoylated proteins, while the lower bands are free SUM O. The lower panel shows a coomassie-stained gel showing total protein as a loading control. Lane 1, wild type; lane 2, rcd1-3; sro1-1; lane 3, nua-1; lane 4, nua-2. (B) Expression of the stress-inducible gene PARP2 is increased in rcd1-3; sro1-1 seedlings. RT -PCR done from two biological replicates with RNA extracted from seedlings is shown. Primers used to amplify PARP2 were as follows: PAR P2RT F (GCA AGC CCA CAT AA G CC G TGG AGG) and PAR P2RTR (TGC CT G CTC TT G AAT TT G TTT AC G TGC). Actin expression was used as a control; primers as in Teotia and Lamb.⁷ Lane 1, wild type; lane 2, rcd1-3; lane 3, sro1-1; lane 4, rcd1-3; sro1-1.

In conclusion, we hypothesize that RCD1 and SRO1 are negative regulators of ROS and/or nitric oxide. When their function is compromised, these compounds accumulate, even in the absence of stress conditions. This causes the plant to develop as if under constitutive abiotic stress, leading to a SIMR phenotype even under ideal growth conditions. Further experimentation will be required to test this hypothesis.

Abbreviations

RCD1

RADICAL INDUCED CELL DEATH 1

SRO1

similar to RCD one1

H₂O₂

hydrogen peroxide

ROS

reactive oxygen species

SIMR

stress-induced morphogenetic response

PARP

poly(ADP-ribose) polymerase

NUA

nuclear pore anchor

Addendum to: Teotia S, Lamb RS. The paralogous genes RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 have partially redundant functions during Arabidopsis development. Plant Physiol. 2009;151:180–198. doi: 10.1104/pp.109.142786.