Dysfunctional mitochondria regulate the size of root apical meristem and leaf development in Arabidopsis

Abstract

Mitochondria play an important role in maintaining metabolic and energy homeostasis in the plant cell. Thus, perturbation of mitochondrial structure and function will affect plant growth and development. Arabidopsis slow growth3 (slo3) is defective in At3g61360 that encodes a pentatricopeptide repeat (PPR) protein. Analysis of slo3 mitochondrial RNA metabolism revealed that the splicing of nad7 intron 2 is impaired, which leads to a dramatic reduction in complex I activity. So the SLO3 PPR protein is a splicing factor that is required for the removal of nad7 intron 2 in Arabidopsis. The slo3 mutant plants have obvious phenotypes with severe growth retardation and delayed development. The size of root apical meristem (RAM) is reduced and the production of meristem cells is decreased in slo3. Furthermore, the rosette leaves of slo3 are curled or crinkled, which may be derived from uneven growth of the leaf surface. The underlying mechanisms by which dysfunctional mitochondria affect these growth and developmental phenotypes have yet to be established. Nonetheless, plant hormone auxin is known to play an important role in orchestrating the development of RAM and leaf shape. It is possible that dysfunctional mitochondria may interact with auxin signaling pathways to regulate the boundary of RAM and the cell division arrest front during leaf growth in Arabidopsis.

Pentatricopeptide repeat (PPR) proteins are the largest RNA-binding protein family found in all eukaryotes. These proteins usually contain 2–30 tandem repeats of a degenerate 35 amino acid motif, which folds into a pair of antiparallel helices.¹ The model plant Arabidopsis thaliana contains approximately 450 PPR proteins, which are further divided into P and PLS subfamilies according to the nature of the PPR motifs. Proteins in the P subfamily contain the classic PPR (P) motif, which usually do not have any other conserved domains. Members of the PLS subfamily contain canonical PPR motifs (P) and long (L) or short (S) variant PPR motifs in a pattern of P-L-S. The PLS subfamily is further divided into PLS, E and DYW subclasses based on their C-terminal domains.²

Thus far, most of the characterized plant PPR proteins are involved in RNA metabolism in chloroplasts or mitochondria.³ In general, the P subfamily PPR proteins are involved in RNA cleavage, splicing, stabilization and translation, whereas members of the PLS subfamily are mostly associated with RNA editing in chloroplasts and mitochondria.^3,4 Computational prediction and crystal structure analysis reveal that PPR proteins may bind RNA via a one PPR domain-one nucleotide module.^1,5 Therefore, the diverse array of PPR motifs may constitute a sequence-specific RNA binding protein.

We previously reported that the Arabidopsis slow growth1 (slo1) mutant is defective in a PPR protein of the E subclass.⁶ The SLO1 PPR protein is required for RNA editing of mitochondrial nad4-449 and nad9-328.⁶ Recently, we reported the characterization of another Arabidopsis slo mutant, slo3, which is similar to slo1 in growth phenotypes.⁷ The SLO3 gene encodes a PPR protein of the P subfamily that is required for the splicing of nad7 intron 2 in Arabidopsis mitochondria.⁷ The respiratory complex I activity is dramatically reduced in slo3, which may be responsible for the slow growth phenotypes of the mutant.⁷ Although these molecular defects are sufficient to explain the retarded growth and delayed development in the slo3 mutant, still, how exactly dysfunctional mitochondria affect plant growth and development remain unclear.

It is comprehensible that energy production and redox status will be compromised in plants containing dysfunctional mitochondria. It has been proposed that these defects may result in a shorter meristem zone (MZ) and reduced cell division rate in the primary root.⁸ We used ethynyl deoxyuridine (EdU) staining to demonstrate that the slo3 mutant has significantly fewer proliferating cells than wild type (WT) in the primary root.⁷ Since the modified nucleoside EdU is incorporated during DNA synthesis, those EdU-stained cells may represent dividing cells in the root apical meristem (RAM) during the treatment. Thus, these results suggest that the slow growth of slo3 is due to a reduction in meristem size and a decrease in cell production rate in the primary root.⁷

The rate of cell production in a root meristem is proportional to the number of dividing cells times cell division rate. It is suggested that cell division rate in the root meristem rarely changes.⁹ By contrast, changes in meristem length by setting the boundaries for meristem and elongation zone play a key regulatory role in root growth.¹⁰ We used EdU to stain the primary roots of WT and slo3 for 30 min and consistently found that the density of EdU-stained cells in the MZ was similar between WT and slo3 (Fig. 1). These results imply that the cell division rate is not significantly reduced in the primary root of slo3. The decrease in cell production rate in the mutant root may have resulted from a low number of meristematic cells, which divide at the same rate as those of the wild type. This observation is consistent with the notion that cell division in the root MZ is at a constant rate.⁹ It is the boundary setting between meristem and elongation zone (e.g. transition zone, TZ) rather than the cell division rate in MZ that plays a major role in the control of root growth.¹⁰

Figure 1.

The density of ethynyl deoxyuridine (EdU)-stained cells is similar in the primary roots of wild-type (WT) and slo3 seedlings. Roots of 7-day-old Arabidopsis seedlings were incubated in growth medium containing EdU for 30 min and observed under confocal microscope. The green fluorescent EdU signals represent newly synthesized DNA. The slo3 mutant root has fewer proliferating cells, but the proliferation rate is not significantly reduced compared with that of the wild type.

It is not clear how dysfunctional mitochondria can affect the length of MZ in the slo3 mutant. The formation of RAM depends on the specification of a stem cell niche and the development of quiescent center (QC).^11-13 Auxin-associated redox regulation is involved in the establishment and maintenance of QC.¹⁴ Interestingly, mitochondria have been shown to play an important role in the regulation of redox homeostasis in the QC.¹⁵ It is possible that dysfunctional mitochondria in the slo3 root may affect RAM via auxin-associated redox regulation in the QC. In addition to the establishment of QC and stem cell niche, auxin transport and distribution are important for determining root meristem size in Arabidopsis.^11,16 Increasing evidence suggests that reactive oxygen species (ROS) will affect auxin homeostasis and signaling.¹⁷ Changes in ROS levels may perturb auxin functions and affect the size of RAM. It is known that the functional state of mitochondria will affect the expression of nuclear genes via retrograde signaling.¹⁸ Interestingly, one of the mitochondrial retrograde regulated genes is directly involved in auxin homeostasis.^19,20 It is possible that dysfunctional mitochondria leading to changes in ROS levels will indirectly affect auxin homeostasis and signaling, which in turn regulates the size of RAM in the slo3 mutant.

Alternatively, ROS may have direct effects on RAM size independent of auxin. ROS plays an important role in regulating cell proliferation in animals.²¹ It has been shown that ROS also controls transition from proliferation to differentiation in the Arabidopsis root.²² The interaction between plant hormones auxin and cytokinin plays a key role in controlling Arabidopsis root meristem size.^{11-13, 23-25} The Arabidopsis UPBEAT1 (UPB1) transcription factor regulates ROS distribution and thus controls the proliferation status of the cells in the root tip.²² Interestingly, the UPB1 regulation of root growth is independent of the auxin/cytokinin signaling pathway.²² It is possible that changes in ROS levels derived from dysfunctional mitochondria may directly regulate the size of RAM by controlling the transition from proliferation to differentiation via an auxin-independent pathway.

In addition to roots, cell proliferation in leaves may be also perturbed in the slo3 mutant. While the wild-type Arabidopsis normally takes about 4 weeks to develop rosette leaves and form an inflorescent stem, the slo3 mutant requires approximately 6 weeks to have fully developed rosette leaves under a 16 h light/8 h dark cycle (Fig. 2). In addition to delayed development, the slo3 rosette leaves are usually curly, wavy or crinkled (Fig. 2). This leaf phenotype is reminiscent of the cincinnata (cin)-like tcp and auxin homeostasis mutants in Arabidopsis.^26-28 In cin-like tcp mutants, the distribution of growth in a leaf is perturbed, which results in a wavy-leaf phenotype.²⁶ CIN-like TCP transcription factors act upstream of the auxin signaling pathway to regulate differentiation of leaves in Arabidopsis.²⁷ Thus, auxin plays a pivotal role in sculpting the final leaf shape. It is conceivable that dysfunctional mitochondria may perturb auxin homeostasis and/or signaling, which in turn affects the development of rosette leaves.

Figure 2.

The slo3 mutant has curly rosette leaves. In wild-type (WT) Arabidopsis, 4-week-old rosette leaves are flat (left, WT). The slo3 mutant is delayed in development and takes approximately 6 weeks to bolt. The rosette leaves of 6-week-old slo3 mutants are curly or crinkled (slo3). Top- and side-view of 2 independent slo3 plants are shown in the middle and right panel, respectively.

Mitochondria play an important role in energy production, redox regulation, metabolic homeostasis, and signaling in plants. Most mitochondrial mutants have reduced growth rates and have a short root phenotype. Interesting, many Arabidopsis mitochondrial mutants also have irregular leaf shapes, which are often described as “curly leaves” in the literature.^29-37 Nevertheless, the relationship between dysfunctional mitochondria and curly rosette leaves is rarely discussed or explored. One of the common themes for root and leaf growth is the regulation of cell proliferation, which includes setting the boundaries for meristem and elongation zone in the root, and control of the cell division arrest front in the leaf. Mitochondria may play an important role in the regulation of these boundaries. Recent studies have implicated that mitochondrial perturbation will negatively affect auxin signaling.²⁰ Thus, retarded root growth and curly rosette leaves observed in slo3 and other mitochondrial mutants may be explained by the interactions between dysfunctional mitochondria and auxin signaling. It will be interesting to further investigate how a perturbation in mitochondrial function may affect the actions of plant hormones, especially auxin homeostasis and signaling, to regulate plant growth and development.

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Funding

This work was supported by grants from the Ministry of Science and Technology and Academia Sinica (98-CDA-L04).