BACKGROUND
as sessile organisms, plants must be able to perceive and adapt to changing environmental cues such as stress conditions.
stresses can be present in combinations, such as the simultaneous heat and drought, or a combination of abiotic and biotic stresses, and can be highly detrimental to plant growth  <cit> .
under such circumstances, plant responses are often non-additive compared to the individual stresses, suggesting a complex regulatory network with significant crosstalk  <cit> .
understanding these regulatory networks, their signaling components, and the genes that influence them are therefore important topics in plant biology.
because plastids are required for photosynthesis, the production of major phytohormones, and other metabolic processes, they are in a key position to sense alterations in the environment and communicate accordingly to the nucleus  <cit> .
for example, light quality affects the plastid transcriptome while light quantity affects plastid signaling, and both influence photomorphogenesis  <cit> .
plastids also play a role in tolerance and signaling against drought, freezing, heat, and oxidative stress  <cit> .
furthermore, disruption of regulators of plastid function, such as pentatricopeptide repeat  proteins, can lead to defects in growth, embryo development, photosynthesis, and leaf pigmentation, among others  <cit> .
as such, the status of plastids has a profound level of control over the entire plant, including its responses to environmental and cellular stress.
msh <dig>  previously known as chm <dig>  is a plant-specific, nuclear-encoded muts homolog that is targeted to both plastids and mitochondria.
loss of msh <dig> causes an array of phenotypes, including variegation, dwarfism, altered leaf morphology, delayed flowering, and male sterility .
additional phenotypes are environmentally-dependent, such as secondary stem growth and aerial rosette formation under short-day conditions  <cit> .
from msh1-suppressed rnai lines in sorghum, pearl millet, tomato, tobacco, and soybean, it is apparent that many of these phenotypes are conserved between monocots and eudicots .
in addition, manipulation of msh <dig> is associated with tolerance to heat, high light, and drought  <cit> , particularly in msh1-depleted plants showing strong developmental phenotypes.
indeed, msh <dig> transcript levels are endogenously down-regulated during stress  <cit> , leading to the possibility that in msh <dig> mutants, stress responses are triggered to cause growth suppression and other phenotypes.
a further consequence of msh <dig> loss is epigenetic, with evidence first appearing from the segregation of msh <dig> rnai plants maintained for multiple generations as hemizygotes.
a proportion of subsequent wild-type segregant progeny lacking the rnai transgene still retained altered growth and delayed flowering phenotypes, which were not cytoplasmically heritable  <cit> .
furthermore, whole genome bisulfite-sequencing of msh <dig> t-dna mutants revealed numerous changes in dna methylation over both gene bodies and transposable elements  <cit> .
in plants, one role of dna methylation is used to silence transposable elements, which can become activated in during stress conditions  <cit> .
in some cases, changes in dna methylation have also been associated with stress-induced gene regulation, such as during phosphate starvation or pseudomonas syringae infection  <cit> , and may also provide the mechanisms basis for stress priming and memory  <cit> .
during propagation of the msh <dig> t-dna materials, we observed that first-generation homozygous msh <dig> mutants  had either no phenotype or only slight variegation, whereas second-generation homozygous msh <dig> mutant  plants displayed the full range of msh1-associated phenotypes  <cit> .
compared to s <dig> generation msh <dig> plants, s <dig> generation msh <dig> plants with mutant phenotypes also had markedly increased amounts of methylation changes in the non-cg context  <cit> .
this contrast raises questions as to what transcriptional changes begin to occur in the msh <dig> -/- s <dig> plants, as opposed to msh <dig> -/- s <dig> plants or later generations.
we hypothesized that the degree of gene expression changes would parallel phenotype and methylome state, distinguishing the transition between the s <dig> and s <dig> generations.
in this study, we performed rna-seq to identify genes that are altered in the first sporophytic generation of msh <dig> loss, as well as those that are induced with the onset of strong phenotypes in the subsequent generation.
using small rna-seq, we also show that mirna profiles and repeat-associated sirna levels change according to msh <dig> generation and phenotype.
together, these data indicate that the variable phenotypes resulting from msh <dig> loss are caused by the triggering of large gene expression networks associated with stress and other pathways, which also ultimately influence genome-wide changes in chromatin organization.
RESULTS
phenotypic and transcriptomic changes from msh <dig> loss build over two generations
we propagated a t-dna insertion line for the msh <dig> locus that contained a mixture of seeds hemizygous and homozygous for the exon insertion .
by self-pollinating a hemizygous msh <dig> +/- plant, we could observe the phenotype and changes occurring in the immediate progeny generation  lacking msh <dig>  as noted in a previous study  <cit> , these msh <dig> -/- s <dig> plants were mild in phenotype , with occasional, slight leaf variegation being the only phenotype observed under normal growth conditions.
in contrast, homozygous msh <dig> -/- plants from generation s <dig> show the range of phenotype seen in chm1- <dig> mutants, such as strong variegation and a wide variation in leaf area , growth rate and flowering time.
from two s <dig> lines, variegation was present in approximately 70% of plants .
as such, mutants showing dwarfism or stunted growth typically, though not necessarily always, also display some form of variegation or chlorotic phenotype.fig.
<dig> phenotypic severity is associated with levels of global transcriptome changes in msh <dig> t-dna mutants.
a representative images of wild-type col- <dig>  msh <dig> -/- s <dig> generation mutants, and msh <dig> -/- s <dig> generation mutants.
b leaf area with means and 95% confidence limits for wild-type col- <dig> , msh <dig> -/- s <dig> , and msh <dig> -/- s <dig> plants .
c pca plot of principal components  <dig> and  <dig> based on log <dig> values.
d venn diagram of differentially expressed genes in msh <dig> mutants compared to msh <dig> +/+ wild-type segregants
to determine what transcriptome changes were associated with particular phenotypes, we performed rna-seq on msh <dig> -/- s <dig> plants, msh <dig> -/- s <dig> variegated plants, and msh <dig> -/- s <dig> variegated & dwarf plants chosen from a single lineage.
msh <dig> +/+ wild-type segregants also derived from the hemizygous t-dna plants were used as the control group.
although there is a possibility that these wild-type segregants may contain subtle dosage-related effects from the previous hemizygous generation, they have no apparent phenotype.
furthermore, they are very closely related in terms of lineage to the msh <dig> mutant individuals, an important advantage for the purposes of this study where small rna changes are also of interest and epigenetics changes may be present  <cit> .
because of the large overall number of gene expression changes observed in msh <dig> mutants, and to minimize the number of potential false positives, we retained only differentially expressed genes  identified by both cuffdiff <dig> and deseq <dig> .
compared against the msh <dig> +/+ wild-type segregant control, we found  <dig> degs in msh <dig> s <dig> plants,  <dig> degs in msh <dig> s <dig> variegated plants, and  <dig> degs in msh <dig> s <dig> variegated & dwarf plants , suggesting that increasing phenotype severity is associated with increased gene expression changes.
from principal component analysis, the msh <dig> s <dig> groups are also more distinct from the wild-type segregants compared to the msh <dig> s <dig> group , and a majority  of the degs in msh <dig> s <dig> variegated plants are also shared with msh <dig> s <dig> variegated & dwarf plants .
despite the relatively small total number of degs in msh <dig> s <dig> plants, over two-thirds of these are also differentially expressed in at least one of the msh <dig> s <dig> groups.
furthermore, of the  <dig> genes that are differentially expressed in all msh <dig> mutants ,  <dig> genes show an intensifying trend between the s <dig> and s <dig> generation , defined by at least a four-fold change.
among these, several are transcription factors, such as scarecrow-like  <dig>  stz, nac <dig>  and dear <dig>  many are also defense-related, including wrky <dig>  wrr <dig>  lurp <dig>  pub <dig>  sdr <dig>  caf1a, and cni <dig> while others are responsive to environmental conditions such as cold  or light .
therefore, despite the lack of a dramatic outward phenotype, several key pathways already appear to be modulated in the msh <dig> -/- s <dig> plants.
on the other hand,  <dig> genes showed a reversal in differential expression direction , including aox1d, the low carbon-induced phi-1/exl <dig>  senescence-associated sag <dig>  the chloroplast-targeted aldo-keto reductase akr4c <dig>  the calcium transporter cax <dig>  and a cold acclimation wcor413-family gene.
an increasing number of studies also indicate that alternative splicing is an important form of regulation in plant development and environmental responses  <cit> .
from our transcriptome data, a gene was considered alternatively spliced if it was identified by cuffdiff2/splicer  <cit>  as having differential isoform expression and by junctionseq  <cit>  as having differential exon or splice junction usage.
using this methodology, there were  <dig>   <dig>  and  <dig> genes with differential isoform expression in the msh <dig> s <dig>  msh <dig> s <dig> variegated, and msh <dig> s <dig> variegated & dwarf plants, respectively .
the majority of isoform-specific events pertained to alternative 5′ or 3′ splice sites, followed closely by alternative transcription start or termination sites .
roughly half of the isoforms  that are differentially expressed in the msh <dig> mutants are also predicted to be sensitive to nonsense-mediated decay , suggesting that these alternative isoforms may be a means to regulate functional transcript abundance  <cit> .
nevertheless, similar to gene expression, there is a trend of subtler isoform-specific changes in the msh <dig> s <dig> generation followed by greater changes in the msh <dig> s <dig> generation.
expansion of differential expression and splicing in specific pathways reflect upon phenotype
we performed enrichment analysis using mapman  <cit>  functional categories on the differentially expressed genes.
several categories were shared between the msh <dig> s <dig> mutants, such as auxin metabolism, stress, photosystem ii components, regulation of wrky transcription factors, and receptor kinase signaling .
cell organization, cell wall modifications, and regulation of bhlh, myb-related, and aux/iaa transcription factor families were particularly enriched in the msh <dig> s <dig> variegated plants, while jasmonate metabolism, calcium signaling, and regulation of constans-like transcription factor families were particularly enriched in the msh <dig> s <dig> variegated and dwarf plants.
constans-like transcription factors are involved in photoperiod, flowering, and circadian rhythm  <cit> , which may be directly related with delayed growth and development in the dwarfed plants.fig.
<dig>
msh <dig> s <dig> mutants share enriched functional categories and changes in general stress gene.
a enrichment of mapman functional categories among differentially expressed genes.
b changes in expression of transcripts and isoforms in multiple stress regulatory genes.
gene-level changes are measured by log <dig> fold-change, while isoform-level changes are measured by the total change in isoform fractions for each gene
among genes with differential isoform expression in msh <dig> s <dig> variegated & dwarf plants, gene ontology  enrichment analysis  <cit>  found  <dig> categories that were enriched, including response to salt stress and response to fructose .
no such enrichment was statistically detectable in msh <dig> s <dig> plants or msh <dig> s <dig> variegated plants, although this is likely affected by the lower overall number of differentially regulated isoforms in those groups.
however, nearly all  of the enriched go categories from analysis of differentially regulated isoforms in msh <dig> s <dig> variegated & dwarf plants were also found in go enrichment analysis of differentially expressed genes in msh <dig> s <dig> mutants , including mapk cascade, regulation of defense response, response to ethylene, and response to cold.
therefore, in addition to differential overall gene expression, alternative isoform regulation and differential splicing likely play a meaningful role in the organismal response to msh <dig> loss.
together, multiple enrichment analyses indicate that abiotic stress and several related pathways are significantly affected in msh <dig> s <dig> mutants of both phenotypic classes.
among  <dig> multiple stress regulatory genes  <cit> ,  <dig> showed differential gene or isoform expression in msh <dig> s <dig> mutants .
additionally,  <dig> drought-responsive genes  <cit>  were differentially expressed in msh <dig> mutants, with  <dig> differentially expressed as early as the msh <dig> s <dig> generation .
differential expression of drought-responsive genes here is consistent with findings of heat and drought tolerance from msh <dig> perturbation  <cit> .
however,  <dig> cold-responsive transcription factors  <cit>  were also differentially expressed in msh <dig> mutants, with  <dig>  up-regulated in the msh <dig> s <dig> generation .
the observed changes across multiple abiotic stress pathways indicate that loss of msh <dig> elicits a general stress response, rather than targeted changes to a specific stress.
triggering of these stress pathways in msh <dig> mutants likely confers the previously observed stress tolerances, but may also be linked to growth rate.
in addition to abiotic stress, the repeated observation of enriched go categories related to biotic stress, such as systemic acquired resistance, regulation of defense response, and regulation of plant-type hypersensitive response is likely related to accumulating evidence of crosstalk between ros signaling and defense responses .
this would be consistent with the enrichment of respiratory burst involved in defense response as early as the s <dig> generation, and sufficient activation of the immune response triggers a decrease in plant growth  <cit> .
as suggested from enrichment analysis, we found that many genes involved in salicylic acid and jasmonate pathways were differentially expressed , as well as other phytohormones such as auxin .
further inspection revealed that a significant number of nbs-lrr genes, the largest family of disease resistance  genes, were also up-regulated .
r genes can be triggered by a variety of environmental changes  <cit> , and may also be particularly labile to changes in dna methylation  <cit> .fig.
<dig>
msh <dig> mutants show triggering of defense responses and changes to plastid and ppr genes.
changes in expression of transcripts and isoforms in a: nbs-lrr genes, b: genes essential for photosynthesis and plastid function, and c: ppr genes
as expected, we also found down-regulation of many genes related to photosynthesis and plastid function, such as light harvesting complex and photosystem i and ii components, as well as up-regulation of several ppr genes of various families .
this includes down-regulation of prin <dig>  which is involved in redox-mediated retrograde signaling and expression of genes transcribed by the plastid-encoded rna polymerase  <cit> , beginning in the msh <dig> s <dig> generation.
gun <dig>  the chlh subunit of mg-chelatase involved in the tetrapyrrole-based retrograde signaling  <cit> , was also down-regulated in msh <dig> -/- s <dig> plants.
previous studies found altered redox levels in msh <dig> mutants, with plastoquinone and phylloquinone biased towards a reduced state  <cit> .
here we also see differential expression of genes related to redox, including the h2o <dig> scavenger apx <dig>  and mdar <dig>  involved in the regeneration of reduced ascorbate .
among nuclear-encoded plastid or mitochondria-associated genes  <cit> , msh <dig> s <dig> variegated plants had a bias towards down-regulation, while in msh <dig> s <dig> variegated & dwarf plants these genes are more evenly split between up-regulation and down-regulation .
however,  <dig> and 40% of the same plastid-associated and mitochondrial-associated degs, respectively, were shared between the two groups .fig.
<dig> ros and circadian rhythm genes are altered in msh <dig> mutants.
changes in expression of transcripts and isoforms in a: ros related genes, and b: circadian rhythm genes
therefore, while one of the initial responses to msh <dig> loss is transcriptional changes in genes directly associated with the organelles, many other changes occur in the nucleus as an additional consequence.
in addition to abiotic and biotic stress pathways previously mentioned, we observed differential expression of several core components of the circadian clock in msh <dig> s <dig> mutants, including lhy and toc <dig> .
in msh <dig> s <dig> variegated & dwarf plants, expression of gi was also altered.
in addition, prr <dig>  a vascular regulator of toc <dig>  <cit> , is differentially expressed beginning in msh <dig> s <dig> generation mutants.
since circadian rhythm is linked to many other gene networks  <cit> , including ros and phytohormone pathways that are altered in msh <dig> mutants, these changes may be an important contributor to the broad pleiotropy in msh <dig> mutants.
rather than an unrelated assortment of pathways, there is accumulating literature indicating that many of those affected in these msh <dig> mutants are interconnected as part of a large signaling network.
for example, organelle perturbation is known to affect ros levels  <cit> , which impacts circadian rhythm and vice versa  <cit> .
in turn, both influence phytohormones , calcium signaling  <cit> , and biotic and abiotic stress responses .
therefore, many of the pathways affected in msh <dig> can be considered as a series of direct consequences from the initial organelle perturbation, with signs of this beginning in the msh <dig> s <dig> generation.
in support of this, comparison of the msh <dig> transcriptome revealed that a number of changes in such pathways were also found in the transcriptomes of plants with other plastid or chloroplast mutations, or chemical disruption of organelle function  .
in msh <dig> mutants, the number of genes within these pathways that are differentially expressed or spliced, and the intensity of their modulation, are associated with increasing phenotypic severity and comprise a distinguishing feature between msh <dig> s <dig>  msh <dig> s <dig> variegated, and msh <dig> s <dig> variegated & dwarf plants.
small rna changes and differential te expression suggest global chromatin alterations in msh <dig> mutants
because micrornas are known to cause large effects in gene regulation and phenotype  <cit> , we also performed small rna-sequencing and aligned  <dig> to 24-nt reads to the genomic sequences of all high-confidence a. thaliana micrornas annotated by mirbase  <cit> .
in msh <dig> s <dig> plants, only mir <dig> was up-regulated compared to the wild-type segregant control, and mir <dig> was also up-regulated in both the msh <dig> s <dig> variegated and msh <dig> s <dig> variegated & dwarf plants .
the mir <dig> regulates the transition from vegetative to reproductive phase, and was previously found to be differentially expressed in chm <dig> mutants  <cit> .
mir <dig>  involved in root architecture and secondary metabolites  <cit> , was also found to be up-regulated in both the msh <dig> s <dig> variegated and msh <dig> s <dig> variegated & dwarf plants, as was mir <dig>  whose function is still unclear.
mir <dig>  which is involved in stress response and nutrient signaling , was down-regulated in both the msh <dig> s <dig> variegated and msh <dig> s <dig> variegated & dwarf plants.
a previous study indicated that the loss of msh <dig> leads to epigenetic changes  <cit> , particularly in phenotypically dwarf plants and with respect to dna methylation in the chg and chh sequence contexts.
chg/chh methylation is primarily associated with heterochromatic regions and transposable elements, and with short interfering rna  production.
small rnas are intimately linked with chromatin organization  <cit> , and believed to play a role in some stress responses, both biotic and abiotic .
we analyzed sirna levels by mapping small rna-sequencing reads to the entire genome using shortstack  <cit> .
most sirna clusters identified belonged to the 24-nt class, particularly those that were differentially expressed , and so were the focus of further analysis.
analogous to what was observed with gene expression changes, msh <dig> s <dig> plants only had  <dig> differentially expressed 24-nt sirna clusters.
in contrast, msh <dig> s <dig> variegated and msh <dig> s <dig> variegated & dwarf plants had  <dig> and  <dig> differentially expressed 24-nt sirna clusters, respectively.
in the msh <dig> s <dig> variegated plants, the majority of these differentially expressed clusters were down-regulated, whereas in the msh <dig> s <dig> variegated & dwarf plants, the majority were up-regulated .fig.
<dig>
msh <dig> mutants have altered 24-nt sirna levels depending on generation and phenotype.
a total number of down-regulated and up-regulated 24-nt sirna clusters in msh <dig> mutants.
b boxplot of distance between differentially expressed 24-nt sirna clusters and the nearest transposable element.
c boxplot of distances between differentially expressed 24-nt sirna clusters and the nearest gene.
d distribution of transposable element superfamilies overlapped by differentially expressed 24-nt sirna clusters, as a proportion within each sample and direction of cluster change
in the msh <dig> s <dig> plants, down-regulated 24-nt sirna clusters are more localized to the pericentromere, whereas up-regulated 24-nt sirna clusters are more spread throughout the chromosome and encompass euchromatic regions .
therefore, down-regulated 24-nt sirna clusters more frequently overlapped transposable elements  and were generally closer to tes than up-regulated 24-nt sirnas were .
however, up-regulated 24-nt sirnas still tended to be located near tes, but were also much closer to genes than down-regulated 24-nt sirnas were.
consistent with this observation, down-regulated 24-nt sirna clusters were mostly associated with gypsy elements, particularly in msh <dig> s <dig> variegated plants, while up-regulated sirna clusters were mostly associated with helitron elements, particularly in msh <dig> s <dig> variegated & dwarf plants .
to a lesser degree, both up-regulated and down-regulated 24-nt sirna clusters were associated with mudr elements across all msh <dig> mutants.
we then evaluated whether any transposable element families showed differential expression in msh <dig> mutants using rna-seq data.
copia and mudr elements showed greatest tendency toward differential expression, regardless of direction of change .
expression of tes did not appear strongly correlated to sirna changes, although this may be masked when evaluating at the superfamily or family level rather than individual elements.
however, in a similar trend to degs and differential sirna clusters, msh <dig> -/- s <dig> variegated & dwarf plants showed the most changes , followed by msh <dig> -/- s <dig> variegated plants , with msh <dig> -/- s <dig> plants having the fewest .
overall, there were more instances of increased te expression than decreased te expression, and two families, athila4c and atmu <dig>  had increased expression in all msh <dig> mutant groups.
furthermore, in msh <dig> s <dig> variegated and dwarf plants, degs were  <dig> -fold enriched within 1 kb of rathe1_cons elements , which were also differentially expressed .
therefore, although not apparently widespread, differential expression of certain transposable elements could be affecting nearby gene expression  <cit> .
we did not find any statistically significant overall enrichment of degs proximal to differentially expressed 24-nt sirna clusters , suggesting that sirna changes here do not impact gene expression at the genome-wide level in an obvious manner.
however, two large gene families, r genes and ppr genes, tend to be associated with repeats and thus may be susceptible to regulation via sirnas  <cit> .
although no enrichment was found for r genes, we observed a  <dig> -fold enrichment for differential expression of ppr genes within 5 kb of a differentially expressed sirna cluster  in msh <dig> s <dig> variegated & dwarf plants, suggesting a possible link between ppr gene expression and proximal sirnas.
these findings of differential sirna and te expression in msh <dig> mutants, coupled with previous studies showing dna methylation changes, indicate that loss of msh <dig> results in broad, global alterations in chromatin organization.
discussion
organelles are essential centers of regulation for multiple cellular signals; seedling, leaf, and flower development; energy and carbon metabolism; and light and temperature adaptation in plants .
loss of the dual organelle-targeted gene msh <dig> triggers changes involving plastid and mitochondria states, resulting in altered ros levels and organellar genome instability, especially within white leaf sectors, and a large reduction in sucrose metabolism .
the resulting variable phenotypes, which requires two generations of homozygosity for complete elaboration but is not amenable to fixation, complicates studies into its function.
here, we performed rna-seq and srna-seq on mutants newly homozygous for msh <dig> t-dna insertion to expand upon the global and pathway-specific changes associated with msh <dig> loss of function, and in particular to each phenotype.
using this segregating msh <dig> t-dna material, we found subtle gene expression changes in the first homozogyous generation related to plastid redox regulation, plant defense, temperature response, and circadian rhythm.
despite this, the first homozygous generation shows little alteration in phenotype, and the total number of changes and the magnitude of the changes are still relatively small .
since msh <dig> expression is particularly high within carpels  <cit> , msh <dig> function may be particularly important during reproduction and the gametophyte stage, where disruption of organelle functions during this stage could have larger consequences on the cell  <cit> .
this would be consistent with the much stronger phenotypes observed by the second sporophytic generation, where gene expression changes expand into additional pathways, including auxin and other phytohormones influencing plant development, along with high induction of stress response pathways such as salt, cold, and oxidative stress.
consistent with enrichment for abiotic and biotic stress transcriptional changes, msh <dig> mutants have been found to be tolerant to high light and heat stress  <cit> .
in plants, repeated stress experience from drought, heat, or pathogens can lead to priming that then facilitates faster future responses within the same generation, and in some cases the underlying mechanism of memory appears to be epigenetic  <cit> .
whole genome bisulfite-sequencing of msh <dig> mutants previously revealed numerous changes in dna methylation over both gene bodies and transposable elements  <cit> , leading to the possibility of epigenetic feedback as a response to msh <dig> loss and heritable methylation changes at stress-responsive loci  <cit> .
however, identification of causative dna methylation changes is hampered by spontaneous differences that are a function of generational distance between individual plants or other stochastic events  <cit> .
this has complicated efforts to determine essential locus-specific methylation signals from msh <dig> loss, but the application of new methods in methylome analysis may aid future investigation  <cit> .
as another approach to epigenetic changes, we examined small rna populations within msh <dig> mutants.
much greater 24-nt sirna changes occurred by the second sporophytic generation of msh <dig> loss than within the first generation, with different preferences in chromosome context  between up-regulated and down-regulated clusters.
however, with the exception of some ppr genes, we did not observe an association between differential sirna levels and gene expression.
thus, although changes in sirnas, te expression, and dna methylation strongly suggest changes to chromatin organization in msh <dig> mutants, these could be broad but not locus-specific effects associated with induction of stress responses.
alternatively, the impact of specific chromatin-level changes upon gene expression may simply be uncommon or difficult to conclusively detect, but are important occurrences  <cit> .
a summary schematic of the downstream effects of msh <dig> perturbation, from organelle perturbation to eventual chromatin-level alterations, is depicted in fig.
<dig> fig.
<dig> overview of gene networks altered and other effects resulting from msh <dig> loss
variegation, chlorosis, or stunted growth phenotypes are also observed in several other plastid or chloroplast mutations  <cit> .
in addition, chemical inhibitors of chloroplast biogenesis or function have also been identified.
we reanalyzed public data from  <dig> such mutants or chemical treatments and compared enriched pathways from differentially expressed genes.
we found that most mutants or chemical treatments also induced some degree of abiotic and biotic stress pathways that were also enriched in the msh <dig> mutant.
this supports the broader concept that organelle function is directly linked to stress responses.
however, the msh <dig> gene has an unusual combination of characteristics, including a plastid nucleoid localization pattern within the epidermis and vascular parenchyma tissue, and a large number of secondary phenotypes  <cit> .
furthermore, to our knowledge, no other organellar gene mutant has been directly assayed for changes in dna methylation  <cit> .
identification of protein interactors with msh <dig> would help place the functional relationship of msh <dig> with other chloroplast proteins and variegation mutants.
future studies will also be important for elucidating the direct importance of stress responses, circadian rhythm, and plastid function in msh <dig> phenotypes, and could reveal previously undescribed loci under epigenetic regulation.
CONCLUSIONS
t-dna mutants of the organellar-targeted gene msh <dig> have a wide variety of phenotypes – with variegation and stunted growth being the most obvious among them – but this is only apparent after two generations of homozygosity.
multiple abiotic stress response pathways, pathogen defense, photosynthesis, cell cycle, circadian rhythm, mapk signaling, and phytohormone regulation are all significantly impacted with far-reaching consequences to development, as the level of their induction differs according with phenotypic severity.
additionally, changes in small rna populations and transposable element activity provide further evidence of alterations at the chromatin organization level.
as msh <dig> mutants have been shown to be tolerant to abiotic stress, these results highlight the connection between environment, organelle function, and nuclear regulation, an interplay that is important for understanding stress biology in plants.

 as sessile organisms, plants must be able to perceive and adapt to changing environmental cues such as stress conditions. stresses can be present in combinations, such as the simultaneous heat and drought, or a combination of abiotic and biotic stresses, and can be highly detrimental to plant growth. under such circumstances, plant responses are often non-additive compared to the individual stresses, suggesting a complex regulatory network with significant crosstalk. understanding these regulatory networks, their signaling components, and the genes that influence them are therefore important topics in plant biology. because plastids are required for photosynthesis, the production of major phytohormones, and other metabolic processes, they are in a key position to sense alterations in the environment and communicate accordingly to the nucleus. for example, light quality affects the plastid transcriptome while light quantity affects plastid signaling, and both influence photomorphogenesis. plastids also play a role in tolerance and signaling against drought, freezing, heat, and oxidative stress. furthermore, disruption of regulators of plastid function, such as pentatricopeptide repeat proteins, can lead to defects in growth, embryo development, photosynthesis, and leaf pigmentation, among others. as such, the status of plastids has a profound level of control over the entire plant, including its responses to environmental and cellular stress. msh previously known as chm is a plant-specific, nuclear-encoded muts homolog that is targeted to both plastids and mitochondria. loss of msh causes an array of phenotypes, including variegation, dwarfism, altered leaf morphology, delayed flowering, and male sterility. additional phenotypes are environmentally-dependent, such as secondary stem growth and aerial rosette formation under short-day conditions. from msh1-suppressed rnai lines in sorghum, pearl millet, tomato, tobacco, and soybean, it is apparent that many of these phenotypes are conserved between monocots and eudicots. in addition, manipulation of msh is associated with tolerance to heat, high light, and drought, particularly in msh1-depleted plants showing strong developmental phenotypes. indeed, msh transcript levels are endogenously down-regulated during stress, leading to the possibility that in msh mutants, stress responses are triggered to cause growth suppression and other phenotypes. a further consequence of msh loss is epigenetic, with evidence first appearing from the segregation of msh rnai plants maintained for multiple generations as hemizygotes. a proportion of subsequent wild-type segregant progeny lacking the rnai transgene still retained altered growth and delayed flowering phenotypes, which were not cytoplasmically heritable. furthermore, whole genome bisulfite-sequencing of msh t-dna mutants revealed numerous changes in dna methylation over both gene bodies and transposable elements. in plants, one role of dna methylation is used to silence transposable elements, which can become activated in during stress conditions. in some cases, changes in dna methylation have also been associated with stress-induced gene regulation, such as during phosphate starvation or pseudomonas syringae infection, and may also provide the mechanisms basis for stress priming and memory. during propagation of the msh t-dna materials, we observed that first-generation homozygous msh mutants had either no phenotype or only slight variegation, whereas second-generation homozygous msh mutant plants displayed the full range of msh1-associated phenotypes. compared to s generation msh plants, s generation msh plants with mutant phenotypes also had markedly increased amounts of methylation changes in the non-cg context. this contrast raises questions as to what transcriptional changes begin to occur in the msh -/- s plants, as opposed to msh -/- s plants or later generations. we hypothesized that the degree of gene expression changes would parallel phenotype and methylome state, distinguishing the transition between the s and s generations. in this study, we performed rna-seq to identify genes that are altered in the first sporophytic generation of msh loss, as well as those that are induced with the onset of strong phenotypes in the subsequent generation. using small rna-seq, we also show that mirna profiles and repeat-associated sirna levels change according to msh generation and phenotype. together, these data indicate that the variable phenotypes resulting from msh loss are caused by the triggering of large gene expression networks associated with stress and other pathways, which also ultimately influence genome-wide changes in chromatin organization.  phenotypic and transcriptomic changes from msh loss build over two generations we propagated a t-dna insertion line for the msh locus that contained a mixture of seeds hemizygous and homozygous for the exon insertion. by self-pollinating a hemizygous msh +/- plant, we could observe the phenotype and changes occurring in the immediate progeny generation lacking msh as noted in a previous study, these msh -/- s plants were mild in phenotype, with occasional, slight leaf variegation being the only phenotype observed under normal growth conditions. in contrast, homozygous msh -/- plants from generation s show the range of phenotype seen in chm1- mutants, such as strong variegation and a wide variation in leaf area, growth rate and flowering time. from two s lines, variegation was present in approximately 70% of plants. as such, mutants showing dwarfism or stunted growth typically, though not necessarily always, also display some form of variegation or chlorotic phenotype.fig. phenotypic severity is associated with levels of global transcriptome changes in msh t-dna mutants. a representative images of wild-type col- msh -/- s generation mutants, and msh -/- s generation mutants. b leaf area with means and 95% confidence limits for wild-type col-, msh -/- s, and msh -/- s plants. c pca plot of principal components and based on log values. d venn diagram of differentially expressed genes in msh mutants compared to msh +/+ wild-type segregants to determine what transcriptome changes were associated with particular phenotypes, we performed rna-seq on msh -/- s plants, msh -/- s variegated plants, and msh -/- s variegated & dwarf plants chosen from a single lineage. msh +/+ wild-type segregants also derived from the hemizygous t-dna plants were used as the control group. although there is a possibility that these wild-type segregants may contain subtle dosage-related effects from the previous hemizygous generation, they have no apparent phenotype. furthermore, they are very closely related in terms of lineage to the msh mutant individuals, an important advantage for the purposes of this study where small rna changes are also of interest and epigenetics changes may be present. because of the large overall number of gene expression changes observed in msh mutants, and to minimize the number of potential false positives, we retained only differentially expressed genes identified by both cuffdiff and deseq. compared against the msh +/+ wild-type segregant control, we found degs in msh s plants, degs in msh s variegated plants, and degs in msh s variegated & dwarf plants, suggesting that increasing phenotype severity is associated with increased gene expression changes. from principal component analysis, the msh s groups are also more distinct from the wild-type segregants compared to the msh s group, and a majority of the degs in msh s variegated plants are also shared with msh s variegated & dwarf plants. despite the relatively small total number of degs in msh s plants, over two-thirds of these are also differentially expressed in at least one of the msh s groups. furthermore, of the genes that are differentially expressed in all msh mutants, genes show an intensifying trend between the s and s generation, defined by at least a four-fold change. among these, several are transcription factors, such as scarecrow-like stz, nac and dear many are also defense-related, including wrky wrr lurp pub sdr caf1a, and cni while others are responsive to environmental conditions such as cold or light. therefore, despite the lack of a dramatic outward phenotype, several key pathways already appear to be modulated in the msh -/- s plants. on the other hand, genes showed a reversal in differential expression direction, including aox1d, the low carbon-induced phi-1/exl senescence-associated sag the chloroplast-targeted aldo-keto reductase akr4c the calcium transporter cax and a cold acclimation wcor413-family gene. an increasing number of studies also indicate that alternative splicing is an important form of regulation in plant development and environmental responses. from our transcriptome data, a gene was considered alternatively spliced if it was identified by cuffdiff2/splicer as having differential isoform expression and by junctionseq as having differential exon or splice junction usage. using this methodology, there were and genes with differential isoform expression in the msh s msh s variegated, and msh s variegated & dwarf plants, respectively. the majority of isoform-specific events pertained to alternative 5′ or 3′ splice sites, followed closely by alternative transcription start or termination sites. roughly half of the isoforms that are differentially expressed in the msh mutants are also predicted to be sensitive to nonsense-mediated decay, suggesting that these alternative isoforms may be a means to regulate functional transcript abundance. nevertheless, similar to gene expression, there is a trend of subtler isoform-specific changes in the msh s generation followed by greater changes in the msh s generation. expansion of differential expression and splicing in specific pathways reflect upon phenotype we performed enrichment analysis using mapman functional categories on the differentially expressed genes. several categories were shared between the msh s mutants, such as auxin metabolism, stress, photosystem ii components, regulation of wrky transcription factors, and receptor kinase signaling. cell organization, cell wall modifications, and regulation of bhlh, myb-related, and aux/iaa transcription factor families were particularly enriched in the msh s variegated plants, while jasmonate metabolism, calcium signaling, and regulation of constans-like transcription factor families were particularly enriched in the msh s variegated and dwarf plants. constans-like transcription factors are involved in photoperiod, flowering, and circadian rhythm, which may be directly related with delayed growth and development in the dwarfed plants.fig. msh s mutants share enriched functional categories and changes in general stress gene. a enrichment of mapman functional categories among differentially expressed genes. b changes in expression of transcripts and isoforms in multiple stress regulatory genes. gene-level changes are measured by log fold-change, while isoform-level changes are measured by the total change in isoform fractions for each gene among genes with differential isoform expression in msh s variegated & dwarf plants, gene ontology enrichment analysis found categories that were enriched, including response to salt stress and response to fructose. no such enrichment was statistically detectable in msh s plants or msh s variegated plants, although this is likely affected by the lower overall number of differentially regulated isoforms in those groups. however, nearly all of the enriched go categories from analysis of differentially regulated isoforms in msh s variegated & dwarf plants were also found in go enrichment analysis of differentially expressed genes in msh s mutants, including mapk cascade, regulation of defense response, response to ethylene, and response to cold. therefore, in addition to differential overall gene expression, alternative isoform regulation and differential splicing likely play a meaningful role in the organismal response to msh loss. together, multiple enrichment analyses indicate that abiotic stress and several related pathways are significantly affected in msh s mutants of both phenotypic classes. among multiple stress regulatory genes, showed differential gene or isoform expression in msh s mutants. additionally, drought-responsive genes were differentially expressed in msh mutants, with differentially expressed as early as the msh s generation. differential expression of drought-responsive genes here is consistent with findings of heat and drought tolerance from msh perturbation. however, cold-responsive transcription factors were also differentially expressed in msh mutants, with up-regulated in the msh s generation. the observed changes across multiple abiotic stress pathways indicate that loss of msh elicits a general stress response, rather than targeted changes to a specific stress. triggering of these stress pathways in msh mutants likely confers the previously observed stress tolerances, but may also be linked to growth rate. in addition to abiotic stress, the repeated observation of enriched go categories related to biotic stress, such as systemic acquired resistance, regulation of defense response, and regulation of plant-type hypersensitive response is likely related to accumulating evidence of crosstalk between ros signaling and defense responses. this would be consistent with the enrichment of respiratory burst involved in defense response as early as the s generation, and sufficient activation of the immune response triggers a decrease in plant growth. as suggested from enrichment analysis, we found that many genes involved in salicylic acid and jasmonate pathways were differentially expressed, as well as other phytohormones such as auxin. further inspection revealed that a significant number of nbs-lrr genes, the largest family of disease resistance genes, were also up-regulated. r genes can be triggered by a variety of environmental changes, and may also be particularly labile to changes in dna methylation.fig. msh mutants show triggering of defense responses and changes to plastid and ppr genes. changes in expression of transcripts and isoforms in a: nbs-lrr genes, b: genes essential for photosynthesis and plastid function, and c: ppr genes as expected, we also found down-regulation of many genes related to photosynthesis and plastid function, such as light harvesting complex and photosystem i and ii components, as well as up-regulation of several ppr genes of various families. this includes down-regulation of prin which is involved in redox-mediated retrograde signaling and expression of genes transcribed by the plastid-encoded rna polymerase, beginning in the msh s generation. gun the chlh subunit of mg-chelatase involved in the tetrapyrrole-based retrograde signaling, was also down-regulated in msh -/- s plants. previous studies found altered redox levels in msh mutants, with plastoquinone and phylloquinone biased towards a reduced state. here we also see differential expression of genes related to redox, including the h2o scavenger apx and mdar involved in the regeneration of reduced ascorbate. among nuclear-encoded plastid or mitochondria-associated genes, msh s variegated plants had a bias towards down-regulation, while in msh s variegated & dwarf plants these genes are more evenly split between up-regulation and down-regulation. however, and 40% of the same plastid-associated and mitochondrial-associated degs, respectively, were shared between the two groups.fig. ros and circadian rhythm genes are altered in msh mutants. changes in expression of transcripts and isoforms in a: ros related genes, and b: circadian rhythm genes therefore, while one of the initial responses to msh loss is transcriptional changes in genes directly associated with the organelles, many other changes occur in the nucleus as an additional consequence. in addition to abiotic and biotic stress pathways previously mentioned, we observed differential expression of several core components of the circadian clock in msh s mutants, including lhy and toc. in msh s variegated & dwarf plants, expression of gi was also altered. in addition, prr a vascular regulator of toc, is differentially expressed beginning in msh s generation mutants. since circadian rhythm is linked to many other gene networks, including ros and phytohormone pathways that are altered in msh mutants, these changes may be an important contributor to the broad pleiotropy in msh mutants. rather than an unrelated assortment of pathways, there is accumulating literature indicating that many of those affected in these msh mutants are interconnected as part of a large signaling network. for example, organelle perturbation is known to affect ros levels, which impacts circadian rhythm and vice versa. in turn, both influence phytohormones, calcium signaling, and biotic and abiotic stress responses. therefore, many of the pathways affected in msh can be considered as a series of direct consequences from the initial organelle perturbation, with signs of this beginning in the msh s generation. in support of this, comparison of the msh transcriptome revealed that a number of changes in such pathways were also found in the transcriptomes of plants with other plastid or chloroplast mutations, or chemical disruption of organelle function. in msh mutants, the number of genes within these pathways that are differentially expressed or spliced, and the intensity of their modulation, are associated with increasing phenotypic severity and comprise a distinguishing feature between msh s msh s variegated, and msh s variegated & dwarf plants. small rna changes and differential te expression suggest global chromatin alterations in msh mutants because micrornas are known to cause large effects in gene regulation and phenotype, we also performed small rna-sequencing and aligned to 24-nt reads to the genomic sequences of all high-confidence a. thaliana micrornas annotated by mirbase. in msh s plants, only mir was up-regulated compared to the wild-type segregant control, and mir was also up-regulated in both the msh s variegated and msh s variegated & dwarf plants. the mir regulates the transition from vegetative to reproductive phase, and was previously found to be differentially expressed in chm mutants. mir involved in root architecture and secondary metabolites, was also found to be up-regulated in both the msh s variegated and msh s variegated & dwarf plants, as was mir whose function is still unclear. mir which is involved in stress response and nutrient signaling, was down-regulated in both the msh s variegated and msh s variegated & dwarf plants. a previous study indicated that the loss of msh leads to epigenetic changes, particularly in phenotypically dwarf plants and with respect to dna methylation in the chg and chh sequence contexts. chg/chh methylation is primarily associated with heterochromatic regions and transposable elements, and with short interfering rna production. small rnas are intimately linked with chromatin organization, and believed to play a role in some stress responses, both biotic and abiotic. we analyzed sirna levels by mapping small rna-sequencing reads to the entire genome using shortstack. most sirna clusters identified belonged to the 24-nt class, particularly those that were differentially expressed, and so were the focus of further analysis. analogous to what was observed with gene expression changes, msh s plants only had differentially expressed 24-nt sirna clusters. in contrast, msh s variegated and msh s variegated & dwarf plants had and differentially expressed 24-nt sirna clusters, respectively. in the msh s variegated plants, the majority of these differentially expressed clusters were down-regulated, whereas in the msh s variegated & dwarf plants, the majority were up-regulated.fig. msh mutants have altered 24-nt sirna levels depending on generation and phenotype. a total number of down-regulated and up-regulated 24-nt sirna clusters in msh mutants. b boxplot of distance between differentially expressed 24-nt sirna clusters and the nearest transposable element. c boxplot of distances between differentially expressed 24-nt sirna clusters and the nearest gene. d distribution of transposable element superfamilies overlapped by differentially expressed 24-nt sirna clusters, as a proportion within each sample and direction of cluster change in the msh s plants, down-regulated 24-nt sirna clusters are more localized to the pericentromere, whereas up-regulated 24-nt sirna clusters are more spread throughout the chromosome and encompass euchromatic regions. therefore, down-regulated 24-nt sirna clusters more frequently overlapped transposable elements and were generally closer to tes than up-regulated 24-nt sirnas were. however, up-regulated 24-nt sirnas still tended to be located near tes, but were also much closer to genes than down-regulated 24-nt sirnas were. consistent with this observation, down-regulated 24-nt sirna clusters were mostly associated with gypsy elements, particularly in msh s variegated plants, while up-regulated sirna clusters were mostly associated with helitron elements, particularly in msh s variegated & dwarf plants. to a lesser degree, both up-regulated and down-regulated 24-nt sirna clusters were associated with mudr elements across all msh mutants. we then evaluated whether any transposable element families showed differential expression in msh mutants using rna-seq data. copia and mudr elements showed greatest tendency toward differential expression, regardless of direction of change. expression of tes did not appear strongly correlated to sirna changes, although this may be masked when evaluating at the superfamily or family level rather than individual elements. however, in a similar trend to degs and differential sirna clusters, msh -/- s variegated & dwarf plants showed the most changes, followed by msh -/- s variegated plants, with msh -/- s plants having the fewest. overall, there were more instances of increased te expression than decreased te expression, and two families, athila4c and atmu had increased expression in all msh mutant groups. furthermore, in msh s variegated and dwarf plants, degs were -fold enriched within 1 kb of rathe1_cons elements, which were also differentially expressed. therefore, although not apparently widespread, differential expression of certain transposable elements could be affecting nearby gene expression. we did not find any statistically significant overall enrichment of degs proximal to differentially expressed 24-nt sirna clusters, suggesting that sirna changes here do not impact gene expression at the genome-wide level in an obvious manner. however, two large gene families, r genes and ppr genes, tend to be associated with repeats and thus may be susceptible to regulation via sirnas. although no enrichment was found for r genes, we observed a -fold enrichment for differential expression of ppr genes within 5 kb of a differentially expressed sirna cluster in msh s variegated & dwarf plants, suggesting a possible link between ppr gene expression and proximal sirnas. these findings of differential sirna and te expression in msh mutants, coupled with previous studies showing dna methylation changes, indicate that loss of msh results in broad, global alterations in chromatin organization. discussion organelles are essential centers of regulation for multiple cellular signals; seedling, leaf, and flower development; energy and carbon metabolism; and light and temperature adaptation in plants. loss of the dual organelle-targeted gene msh triggers changes involving plastid and mitochondria states, resulting in altered ros levels and organellar genome instability, especially within white leaf sectors, and a large reduction in sucrose metabolism. the resulting variable phenotypes, which requires two generations of homozygosity for complete elaboration but is not amenable to fixation, complicates studies into its function. here, we performed rna-seq and srna-seq on mutants newly homozygous for msh t-dna insertion to expand upon the global and pathway-specific changes associated with msh loss of function, and in particular to each phenotype. using this segregating msh t-dna material, we found subtle gene expression changes in the first homozogyous generation related to plastid redox regulation, plant defense, temperature response, and circadian rhythm. despite this, the first homozygous generation shows little alteration in phenotype, and the total number of changes and the magnitude of the changes are still relatively small. since msh expression is particularly high within carpels, msh function may be particularly important during reproduction and the gametophyte stage, where disruption of organelle functions during this stage could have larger consequences on the cell. this would be consistent with the much stronger phenotypes observed by the second sporophytic generation, where gene expression changes expand into additional pathways, including auxin and other phytohormones influencing plant development, along with high induction of stress response pathways such as salt, cold, and oxidative stress. consistent with enrichment for abiotic and biotic stress transcriptional changes, msh mutants have been found to be tolerant to high light and heat stress. in plants, repeated stress experience from drought, heat, or pathogens can lead to priming that then facilitates faster future responses within the same generation, and in some cases the underlying mechanism of memory appears to be epigenetic. whole genome bisulfite-sequencing of msh mutants previously revealed numerous changes in dna methylation over both gene bodies and transposable elements, leading to the possibility of epigenetic feedback as a response to msh loss and heritable methylation changes at stress-responsive loci. however, identification of causative dna methylation changes is hampered by spontaneous differences that are a function of generational distance between individual plants or other stochastic events. this has complicated efforts to determine essential locus-specific methylation signals from msh loss, but the application of new methods in methylome analysis may aid future investigation. as another approach to epigenetic changes, we examined small rna populations within msh mutants. much greater 24-nt sirna changes occurred by the second sporophytic generation of msh loss than within the first generation, with different preferences in chromosome context between up-regulated and down-regulated clusters. however, with the exception of some ppr genes, we did not observe an association between differential sirna levels and gene expression. thus, although changes in sirnas, te expression, and dna methylation strongly suggest changes to chromatin organization in msh mutants, these could be broad but not locus-specific effects associated with induction of stress responses. alternatively, the impact of specific chromatin-level changes upon gene expression may simply be uncommon or difficult to conclusively detect, but are important occurrences. a summary schematic of the downstream effects of msh perturbation, from organelle perturbation to eventual chromatin-level alterations, is depicted in fig.  fig. overview of gene networks altered and other effects resulting from msh loss variegation, chlorosis, or stunted growth phenotypes are also observed in several other plastid or chloroplast mutations. in addition, chemical inhibitors of chloroplast biogenesis or function have also been identified. we reanalyzed public data from such mutants or chemical treatments and compared enriched pathways from differentially expressed genes. we found that most mutants or chemical treatments also induced some degree of abiotic and biotic stress pathways that were also enriched in the msh mutant. this supports the broader concept that organelle function is directly linked to stress responses. however, the msh gene has an unusual combination of characteristics, including a plastid nucleoid localization pattern within the epidermis and vascular parenchyma tissue, and a large number of secondary phenotypes. furthermore, to our knowledge, no other organellar gene mutant has been directly assayed for changes in dna methylation. identification of protein interactors with msh would help place the functional relationship of msh with other chloroplast proteins and variegation mutants. future studies will also be important for elucidating the direct importance of stress responses, circadian rhythm, and plastid function in msh phenotypes, and could reveal previously undescribed loci under epigenetic regulation.  t-dna mutants of the organellar-targeted gene msh have a wide variety of phenotypes – with variegation and stunted growth being the most obvious among them – but this is only apparent after two generations of homozygosity. multiple abiotic stress response pathways, pathogen defense, photosynthesis, cell cycle, circadian rhythm, mapk signaling, and phytohormone regulation are all significantly impacted with far-reaching consequences to development, as the level of their induction differs according with phenotypic severity. additionally, changes in small rna populations and transposable element activity provide further evidence of alterations at the chromatin organization level. as msh mutants have been shown to be tolerant to abiotic stress, these results highlight the connection between environment, organelle function, and nuclear regulation, an interplay that is important for understanding stress biology in plants. 
stronger phenotypes have been connected to stress tolerance and epigenetic changes, and in arabidopsis t-dna mutants, two generations of homozygosity with the msh insertion are required before severe phenotypes begin to emerge. using rna-seq, we analyze pathways that are associated with msh loss, including abiotic stresses such as cold response, pathogen defense and immune response, salicylic acid, mapk signaling, and circadian rhythm. in all cases, the magnitude of changes among protein-coding genes, transposable elements, and small rnas increases according to generation and phenotypic severity. this study enforces the recognition of organelles as critical integrators of both internal and external cues, and highlights the relationship between organelle and nuclear regulation in fundamental aspects of plant development and stress signaling. using small rna-seq we further identify mirna changes, and uncover sirna trends that indicate modifications at the chromatin organization level. among these genes, muts homolog is notable for generating an assortment of mutant phenotypes with varying degrees of penetrance and pleiotropy. subtle redox and environment-responsive changes also begin in the first generation, in the absence of strong phenotypes. loss of msh is sufficient to cause large-scale regulatory changes in pathways that have been individually linked to one another, but rarely described all together within a single mutant background. these observations prompted us to examine how msh mutants contrast according to generation and phenotype by profiling their respective transcriptomes and small rna populations.
 proper regulation of nuclear-encoded, organelle-targeted genes is crucial for plastid and mitochondrial function. among these genes, muts homolog is notable for generating an assortment of mutant phenotypes with varying degrees of penetrance and pleiotropy. stronger phenotypes have been connected to stress tolerance and epigenetic changes, and in arabidopsis t-dna mutants, two generations of homozygosity with the msh insertion are required before severe phenotypes begin to emerge. these observations prompted us to examine how msh mutants contrast according to generation and phenotype by profiling their respective transcriptomes and small rna populations.  using rna-seq, we analyze pathways that are associated with msh loss, including abiotic stresses such as cold response, pathogen defense and immune response, salicylic acid, mapk signaling, and circadian rhythm. subtle redox and environment-responsive changes also begin in the first generation, in the absence of strong phenotypes. using small rna-seq we further identify mirna changes, and uncover sirna trends that indicate modifications at the chromatin organization level. in all cases, the magnitude of changes among protein-coding genes, transposable elements, and small rnas increases according to generation and phenotypic severity.  loss of msh is sufficient to cause large-scale regulatory changes in pathways that have been individually linked to one another, but rarely described all together within a single mutant background. this study enforces the recognition of organelles as critical integrators of both internal and external cues, and highlights the relationship between organelle and nuclear regulation in fundamental aspects of plant development and stress signaling. our findings also encourage further investigation into potential connections between organelle state and genome regulation vis-á-vis small rna feedback. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords msh1transcriptomeplastidorganellestresssmall rnahttp://dx.doi.org/ /100000865bill and melinda gates foundationopp1088661issue-copyright-statement© the author 2017 