Comprehensive 3D phenotyping reveals continuous morphological variation across genetically diverse sorghum inflorescences.

●Inflorescence architecture in plants is often complex and challenging to quantify, particularly for inflorescences of cereal grasses. Methods for capturing inflorescence architecture and for analyzing the resulting data are limited to a few easily captured parameters that may miss the rich underlying diversity. ●Here, we apply X-ray computed tomography combined with detailed morphometrics, offering new imaging and computational tools to analyze three-dimensional inflorescence architecture. To show the power of this approach, we focus on the panicles of Sorghum bicolor, which vary extensively in numbers, lengths, and angles of primary branches, as well as the three-dimensional shape, size, and distribution of the seed. ●We imaged and comprehensively evaluated the panicle morphology of 55 sorghum accessions that represent the five botanical races in the most common classification system of the species, defined by genetic data. We used our data to determine the reliability of the morphological characters for assigning specimens to race and found that seed features were particularly informative. ●However, the extensive overlap between botanical races in multivariate trait space indicates that the phenotypic range of each group extends well beyond its overall genetic background, indicating unexpectedly weak correlation between morphology, genetic identity, and domestication history.

An epigenetic breeding system in soybean for increased yield and stability.

Epigenetic variation has been associated with a wide range of adaptive phenotypes in plants, but there exist few direct means for exploiting this variation. RNAi suppression of the plant-specific gene, MutS HOMOLOG1 (MSH1), in multiple plant species produces a range of developmental changes accompanied by modulation of defence, phytohormone and abiotic stress response pathways along with methylome repatterning. This msh1-conditioned developmental reprogramming is retained independent of transgene segregation, giving rise to transgene-null 'memory' effects. An isogenic memory line crossed to wild type produces progeny families displaying increased variation in adaptive traits that respond to selection. This study investigates amenability of the MSH1 system for inducing agronomically valuable epigenetic variation in soybean. We developed MSH1 epi-populations by crossing with msh1-acquired soybean memory lines. Derived soybean epi-lines showed increase in variance for multiple yield-related traits including pods per plant, seed weight and maturity time in both glasshouse and field trials. Selected epi-F2:4 and epi-F2:5 lines showed an increase in seed yield over wild type. By epi-F2:6, we observed a return of MSH1-derived enhanced growth back to wild-type levels. Epi-populations also showed evidence of reduced epitype-by-environment (e × E) interaction, indicating higher yield stability. Transcript profiling of epi-lines identified putative signatures of enhanced growth behaviour across generations. Genes related to cell cycle, abscisic acid biosynthesis and auxin response, particularly SMALL AUXIN UP RNAs (SAURs), were differentially expressed in epi-F2:4 lines that showed increased yield when compared to epi-F2:6 . These data support the potential of MSH1-derived epigenetic variation in plant breeding for enhanced yield and yield stability.

Stress-responsive pathways and small RNA changes distinguish variable developmental phenotypes caused by MSH1 loss.

BACKGROUND: Proper regulation of nuclear-encoded, organelle-targeted genes is crucial for plastid and mitochondrial function. Among these genes, MutS Homolog 1 (MSH1) 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 msh1 insertion are required before severe phenotypes begin to emerge. These observations prompted us to examine how msh1 mutants contrast according to generation and phenotype by profiling their respective transcriptomes and small RNA populations. RESULTS: Using RNA-seq, we analyze pathways that are associated with MSH1 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. CONCLUSION: Loss of MSH1 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.

ENTIRE and GOBLET promote leaflet development in tomato by modulating auxin response.

Compound leaves produce leaflets in a highly controlled yet flexible pattern. Here, we investigate the interaction between auxin, the putative auxin response inhibitor ENTIRE (E, SlIAA9) and the CUC transcription factor GOBLET (GOB) in compound-leaf development in tomato (Solanum lycopersicum). Auxin maxima, monitored by the auxin response sensor DR5, marked and preceded leaflet and lobe initiation. The DR5 signal increased, but maxima were partially retained in response to the external or internal elevation of auxin levels. E directly interacted with the auxin receptors SlTIR1 and SlAFB6. Furthermore, E was stabilized by a mutation in domain II of the protein and by the inhibition of auxin or proteasome activity, implying that E is subjected to auxin-mediated degradation. In e mutants the DR5 signal expanded to include the complete leaf margin, and leaf-specific overexpression of a stabilized form of E inhibited the DR5 signal and lamina expansion. Genetic manipulation of GOB activity altered the distribution of the DR5 signal, and the inhibition of auxin transport or activity suppressed the GOB overexpression phenotype, suggesting that auxin mediates GOB-regulated leaf patterning. Whereas leaves of single e or gob mutants developed only primary leaflets, the downregulation of both E and GOB resulted in the complete abolishment of leaflet initiation, and in a strong DR5 signal throughout the leaf margin. These results suggest that E and GOB modulate auxin response and leaflet morphogenesis via partly redundant pathways, and that proper leaflet initiation and separation requires distinct boundaries between regions of lamina growth and adjacent regions in which growth is inhibited.

Epigenomic plasticity of Arabidopsis msh1 mutants under prolonged cold stress.

Dynamic transcriptional and epigenetic changes enable rapid adaptive benefit to environmental fluctuations. However, the underlying mechanisms and the extent to which this occurs are not well known. MutS Homolog 1 (MSH1) mutants cause heritable developmental phenotypes accompanied by modulation of defense, phytohormone, stress-response, and circadian rhythm genes, as well as heritable changes in DNA methylation patterns. Consistent with gene expression changes, msh1 mutants display enhanced tolerance for abiotic stress including drought and salt stress, while showing increased susceptibility to freezing temperatures. Despite changes in defense and biotic stress-response genes, msh1 mutants showed increasing susceptibility to the bacterial pathogen Pseudomonas syringae. Our results suggest that chronic cold and low light stress (10°C, 150 µmol m-2 s-1) influences non-CG methylation to a greater degree in msh1 mutants compared to wild-type Col-0. Furthermore, CHG changes are more closely pericentromeric, whereas CHH changes are generally more dispersed. This increased variation in non-CG methylation pattern does not significantly affect the msh1-derived enhanced growth behavior after mutants are crossed with isogenic wild type, reiterating the importance of CG methylation changes in msh1-derived enhanced vigor. These results indicate that msh1methylome is hyper-responsive to environmental stress in a manner distinct from the wild-type response, but CG methylation changes are potentially responsible for growth vigor changes in the crossed progeny.

Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth.

Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.

MSH1-induced non-genetic variation provides a source of phenotypic diversity in Sorghum bicolor.

MutS Homolog 1 (MSH1) encodes a plant-specific protein that functions in mitochondria and chloroplasts. We showed previously that disruption or suppression of the MSH1 gene results in a process of developmental reprogramming that is heritable and non-genetic in subsequent generations. In Arabidopsis, this developmental reprogramming process is accompanied by striking changes in gene expression of organellar and stress response genes. This developmentally reprogrammed state, when used in crossing, results in a range of variation for plant growth potential. Here we investigate the implications of MSH1 modulation in a crop species. We found that MSH1-mediated phenotypic variation in Sorghum bicolor is heritable and potentially valuable for crop breeding. We observed phenotypic variation for grain yield, plant height, flowering time, panicle architecture, and above-ground biomass. Focusing on grain yield and plant height, we found some lines that appeared to respond to selection. Based on amenability of this system to implementation in a range of crops, and the scope of phenotypic variation that is derived, our results suggest that MSH1 suppression provides a novel approach for breeding in crops.

Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots.

The ARP2/3 complex, a highly conserved nucleator of F-actin polymerization, and its activator, the SCAR complex, have been shown to play important roles in leaf epidermal cell morphogenesis in Arabidopsis. However, the intracellular site(s) and function(s) of SCAR and ARP2/3 complex-dependent actin polymerization in plant cells remain unclear. We demonstrate that putative SCAR complex subunits BRK1 and SCAR1 are localized to the plasma membrane at sites of cell growth and wall deposition in expanding cells of leaves and roots. BRK1 localization is SCAR-dependent, providing further evidence of an association between these proteins in vivo. Consistent with plasma membrane localization of SCAR complex subunits, cortical F-actin accumulation in root tip cells is reduced in brk1 mutants. Moreover, mutations disrupting the SCAR or ARP2/3 complex reduce the growth rate of roots and their ability to penetrate semi-solid medium, suggesting reduced rigidity. Cell walls of mutant roots exhibit abnormal structure and composition at intercellular junctions where BRK1 and SCAR1 are enriched in the adjacent plasma membrane. Taken together, our results suggest that SCAR and ARP2/3 complex-dependent actin polymerization promotes processes at the plasma membrane that are important for normal growth and wall assembly.