The chloroplast triggers developmental reprogramming when MUTS HOMOLOG1 is suppressed in plants

A1 and Samples observed with . estimated analyzed with


Introduction
Plants display a surprising capacity for rapid adaptation. Phenotypic response to environmental change is thought to include non-genetic, transgenerational processes (Bonduriansky and Day 2009) that likely integrate epigenetic and/or maternal factors (Danchin et al. 2011;Johannes et al. 2008). Although maternal effects have been shown to influence these plant responses (Galloway, 2005), involvement of organellar processes has not been formally demonstrated.
The MutS HOMOLOG1 (MSH1) gene is unique to plants, and encodes a homolog to the bacterial mismatch repair protein MutS (Abdelnoor et al. 2003) with at least two important changes within the plant lineage during its evolution. The first involved a carboxy-terminal fusion to a GIY-YIG homing endonuclease domain (VI) (Abdelnoor et al. 2006) , and the second inclusion of hydrophobic stretches within the region linking essential DNA binding (I) and ATPase (V) domains. These and other sequence features distinguish the plant protein from any other MutS homolog yet identified (Ogota et al. 2011), and presumably confer unique functional properties.
The MSH1 protein is dual-targeting, localized to both mitochondrial and chloroplast nucleoids (Xu et al. 2011). Disruption of MSH1 enhances recombination at 47 pairs of repeated sequences in the mitochondrial genome of Arabidopsis (Shedge et al. 2007;Arrieta-Montiel et al. 2009;Davila et al. 2011), and gives rise to cytoplasmic male sterility in tomato and tobacco (Sandhu et al. 2007). Within the chloroplast, disruption of MSH1 results in low frequency DNA rearrangements mediated by recombination, together with altered redox properties of the cell and variegation of the plant (Xu et al. 2011). In both cytoplasmic male sterility and variegation, the altered phenotype appears to derive from organellar genomic rearrangement, displaying subsequent maternal inheritance with incomplete penetrance.
Here, we have carried out cross-species comparative studies of MSH1 suppression to investigate phenotypic changes that occur in response to organellar perturbation but do not appear attributable to organellar genome instability. These studies produced evidence of developmental reprogramming in response to chloroplast signals that accompany MSH1 suppression. Remarkably, these developmental changes, once effected, are stable, heritable and independent of the RNAi transgene in subsequent generations, suggesting that these organellar signals influence epigenetic properties of the plant.

MSH1 suppression has similar phenotypic effects in multiple plant species
Whereas MSH1 was originally identified in Arabidopsis, subsequent studies of its properties were investigated by RNAi suppression of MSH1 orthologs in other plant species, including the monocots sorghum and millet, and the dicots soybean, tobacco and tomato. Comparative analysis of MSH1 depletion in these lines produced similar phenotypic changes beyond male sterility and variegation in each species, involving dwarfed growth and reduced internode elongation, enhanced branching, altered leaf morphology, extended juvenility and delayed flowering, as shown in Figure 1. In tobacco, the most pronounced features of the MSH1-dr growth included a dramatic range of altered leaf morphologies, ranging from extremely large and rounded to very narrow and pointed in shape. Delays in tobacco flowering included plants that never flowered and continued to grow vegetatively. Many plants showed extensive alteration in branching pattern (Figure 1). In soybean, leaf morphology was the most pronounced effect in the dwarfed plants, with leaf wrinkling that resembled virus infection. These plants also showed dramatic delays in flowering.
In sorghum, where phenotypic variation was analyzed in detail, MSH1 suppression produced dramatic changes in plant tillering, height, internode elongation and stomatal density (Supplementary Figure 1, Supplementary Table 2). These phenotypic changes were characterized as developmental reprogramming (MSH1-dr), given their cross-species reproducibility and influence on numerous aspects of development.
Phenotypic sorting allowed discrimination of independently sorting and separable developmental changes: variegation, male sterility, and the dwarfed/tillered/delayed flowering phenotypes. The dwarf phenotype was consistently co-inherited with enhanced tillering/altered branching and flowering delay in all plant species, with the phenotype appearing in approximately 20% of the original, unselected msh1 population in Arabidopsis. The phenotype appeared in about 40% of the T3 families in sorghum at an average frequency of 20% in those families in which it was observed (Supplemental Table 1). Because our initial focus for these studies was on documenting the male-sterility phenotype, we did not characterize the exact frequency of the MSH1-dr phenotype in early generations; our detailed characterization of the phenotype was initiated in the T 3 generation.
In the Arabidopsis msh1 mutant, plants showed a range in the severity of the altered phenotype, with ca. 20% clearly dwarfed and 100% delayed in flowering. Selection for the dwarfed plants effectively shifted the resulting population to ca.80% dwarfed, delayed flowering type ( Figure 2A, Table 2A) that, under 10-hour day length, displayed perennial growth features, including aerial rosettes, dramatic elongation of lifespan and enhanced secondary growth of the stem ( Figure 2). This delay in flowering was associated with delay in maturity transition, evident in leaf shape ( Figure 2F). The dwarf msh1 mutant phenotype in Arabidopsis showed partial reversal with GA application ( Figure 2G, Table 2B).

The altered growth patterns are retained following segregation of the RNAi transgene
In sorghum, individuals displaying the MSH1-dr phenotype gave rise to progeny populations fully penetrant for the phenotype (100% dwarfed/enhanced tillering/delayed flowering). Upon segregation of the RNAi transgene in a hemizygous plant displaying the MSH1-dr phenotype, the phenotype was again fully penetrant in the progeny population, regardless of transgene segregation. These observations permitted the development of sorghum lines, devoid of the RNAi transgene, that bred true for the MSH1-dr phenotype over multiple cycles of selfpollination, with six generations confirmed to date (Table 1). The altered phenotype could also be partially reversed by spraying the leaves with 2250 ppm gibberellic acid ( Figure 1C).
Although genetic segregation for the RNAi transgene did not reverse the altered dwarf phenotype in sorghum, non-transgenic segregants displayed slight changes in flowering.
Transgenic plants were nonflowering unless treated with GA; non-transgenic plants were delayed in flowering but did not require GA treatment. In non-transgenic plants, MSH1 transcript levels and MSH1 DNA methylation pattern reverted to wildtype levels (  Table 3). Thus, MSH1 modulation appears to condition changes within the plant that are heritable through self-pollination but reversed through crossing to wildtype. Identical reciprocal crossing results showing reversal of phenotypes imply that these heritable changes are not organellar. In the subsequent F 2 generation, we also observed no evidence of the dwarf phenotype as would be expected if the trait were conditioned by a single recessive locus that was segregating (data not shown).

MSH1 suppression alters numerous plant pathways
Transcript profiling and RT-PCR experiments in the Arabidopsis msh1 mutant identified several nuclear gene expression changes underlying the altered growth types (Table 3). Pathways associated with dwarfing include cell cycle regulation and increased GA catabolism (Table 2B,  Arabidopsis, this alteration was most evident in the depletion of sucrose to undetectable levels. Metabolic priming for environmental stress in sorghum may be evident in the 1.2 to 5.7-fold elevation of sugar and sugar-alcohol levels, an effect that stabilizes osmotic pressure in response to stresses like drought (Ingram and Bartels 1996). Anti-oxidants ascorbate and alphatocopherols were increased, together with the stress-responsive flavones apigenin, apigenin-7-oglucoside, isovitexin, kaempferol 3-O-beta-glucoside, luteolin-7-O-glucoside and vitexin. In Arabidopsis, the response included an increase in oxidized glutathione, as well as sinapate, likely signaling induction of the phenypropanoid pathway, together with the polyamines 1, 3diaminopropane, putrescine and spermidine, which likely influence both stress tolerance and the observed delay in maturity transition (Gill and Tuteja 2010).

The observed developmental reprogramming is the consequence of chloroplast changes
Although several identifiable and intersecting nuclear gene expression networks are altered in the phenotypic variants, MSH1 is an organellar protein. We used genetic hemi-complementation to discriminate between mitochondrial and plastidial influences on msh1-associated phenotypy.
Hemi-complementation lines were developed in Arabidopsis by transgenic introduction of a mitochondrial versus plastid-targeted form of MSH1 to an msh1 mutant as described previously

Discussion
The results we present, suggesting chloroplast influence on multiple growth parameters, are not entirely surprising; GA biosynthesis, light response and vernalization pathways involve chloroplast processes. Mutation of the CND41 gene in tobacco, encoding a chloroplast nucleoid protein with protease activity, can result in reduction of GA 1 levels and a dwarf phenotype What is surprising in MSH1 depletion is not simply the array of phenotypes that emerge, but the programmed and heritable manner in which these intersecting nuclear gene networks respond to organelle perturbation. Numerous genetic mutations are shown to alter chloroplast functions, many producing variegation phenotypes (Sakamoto, 2003;Yu et al. 2007). Yet, no association has been reported of these mutations with similar, developmental reprogramming, implying that a specificity of function rather than general organellar perturbation conditions the msh1 changes.
The hemi-complementation assay was designed to not only discriminate between mitochondrial and plastid contribution the derived phenotype, but to assess whether MSH1 might also function within the nucleus. We observe co-inheritance of variation in flowering time, plant growth rate, branching patterns, stomatal density changes and maturity transition in Arabidopsis and sorghum.
Phenotypic variation for these quantitative traits has been the subject of ecological association mapping studies to understand genotype by environment interactions and plant adaptation in natural environments (Bergelson and Roux 2010). Our results suggest that epigenetic, or "soft" inheritance, processes may support a coordinate modulation of all of these traits in response to environmental cues.

Plant materials and growth conditions
Arabidopsis For GA treatment, 3 week old Arabidopsis plants were treated with 100 µM GA3 twice a week for 3 weeks. Sorghum plants were treated twice with 2500 ppm GA3 starting prior to transition to reproduction, with treatments two weeks apart.
For studies of metabolism and transcript levels, Arabidopsis plant staging was carried out based on leaf number, and plants of same age were used for all experiments. The msh1 mutants are considerably smaller than wildtype at the same age, determined as days after germination.
Plant sampling stage was just before bolting. For sorghum analysis, plants were taken at the 5 to 6-leaf stage. All plants were grown under controlled growth room conditions.

Microscopy
The autofluorescence images of secondary growth were produced with fresh, hand-sectioned

RNA Isolation and Real-Time PCR Analysis
Total Arabidopsis and sorghum RNA was extracted from above-ground tissues of wild-type and mutant or RNAi plants using TRIzol (Invitrogen) extraction procedure followed by purification on RNeasy columns (Qiagen). cDNA was synthesized with SuperScriptIII first-strand synthesis SuperMix for qRT-PCR (Invitrogen). Quantitative PCR was performed on the iCycler iQ system (Biorad) with SYBR GreenER Supermix (Invitrogen). PCR primers are listed in Supplementary

Microarry experiments
Microarray experiments were carried out as described in Xu et al.  Table 1. Inheritance of the dwarf phenotype in T 3 , T 4 and T 5 generations following initial selection of the MSH1-dr (dwarf, high tillering, delayed flowering, non-transgenic) lines in T 2 . Following selection for the MSH1-dr phenotype, all plants showed the dwarf trait in each generation, and these also showed enhanced tillering and delayed flowering, so plant height was used as the measure. Although only three generations are shown, stable heritability of the phenotype has been observed over six generations. All T 3 , T 4 and T 5 plants were significantly lower in plant height than wildtype TX430 (P<0.001). Lack of the MSH1-RNAi transgene was confirmed in all populations by PCR (Supplementary Figure 2). The first two letters of each line designate the generation, the remainder is an in-lab designator for the family.       Consequently, we assume that this elevated level of MSH1 segregating within the T4 generation does not noticeably influence phenotype.