Characterization, comparison, and phylogenetic analyses of chloroplast genomes of Euphorbia species.

The genus Euphorbia (Euphorbiaceae) has near-cosmopolitan distribution and serves as a significant resource for both ornamental and medicinal purposes. Despite its economic importance, Euphorbia's taxonomy has long been challenged by the intricate nature of morphological traits exhibiting high levels of convergence. While molecular markers are essential for phylogenetic studies, their availability for Euphorbia has been limited. To address this gap, we conducted comparative analyses focusing on the chloroplast (CP) genomes of nine Euphorbia species, incorporating three newly sequenced and annotated accessions. In addition, phylogenetic informativeness and nucleotide diversity were computed to identify candidate markers for phylogenetic analyses among closely related taxa in the genus. Our investigation revealed relatively conserved sizes and structures of CP genomes across the studied species, with notable interspecific variations observed primarily in non-coding regions and IR/SC borders. By leveraging phylogenetic informativeness and nucleotide diversity, we identified rpoB gene as the optimal candidate for species delimitation and shallow-level phylogenetic inference within the genus. Through this comprehensive analysis of CP genomes across multiple taxa, our study sheds light on the evolutionary dynamics and taxonomic intricacies of Euphorbia, offering valuable insights into its CP genome evolution and taxonomy.

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

The chloroplast genome of a subtropical tree Sassafras randaiense (Hayata) Rehder, 1920 (Lauraceae).

The complete chloroplast genome sequence of Sassafras randaiense (Hayata) Rehder, 1920, a subtropical tree in the family Lauraceae, was determined. For a better understanding of the differences between S. randaiense and S. tzumu, the complete chloroplast genome of S. randaiense was sequenced and analyzed. The complete chloroplast genome is 151,781 bp in length, consisting of a pair of inverted repeat (IR) regions of 20,114 bp, one large single-copy (LSC) region of 92,740 bp, and one small single-copy (SSC) region of 18,813 bp. The overall GC content of the complete chloroplast genome is 39.2%. Further, maximum-likelihood phylogenetic analysis was conducted using 31 complete plastome sequences, which support that S. randaiense and S. tzumu are nested among the members of Cinnamomum, suggesting that Sassafras belongs to Cinnamomum.

Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts.

Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 µg/mL luciferase in batch reactions and 60 ± 4 µg/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

Complete chloroplast genome and phylogenetic analysis of Amorphophallus paeoniifolius (Araceae).

Amorphophallus paeoniifolius (Dennst.) Nicolson, 1885, often known as elephant foot yam, is a tropical tuber crop that originates from south-east Asia and belongs to the Araceae family. It is known for its high production potential and popularity as a medicinal plant. However, the phylogeny and genes for this species are still unavailable. In this study, the first complete chloroplast genome of A. paeoniifolius was reported and phylogenetic analysis was conducted with Araceae species. The chloroplast genome was 176,258 bp in length with 34.80% overall GC content and includes a large single-copy (LSC) region (93,951 bp), a small single-copy (SSC) region (15,013 bp), and a pair of inverted repeat (IRs) regions (33,647 bp). The chloroplast genome has 130 genes, which include 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. A maximum-likelihood (ML) phylogenetic analysis indicated that all Amorphophallus species formed a single monophyletic clade with a high bootstrap value and A. paeoniifolius was closely related to A. konjac, A. albus, A. krausei, and A. titanum. The chloroplast genome reported in this study will be useful for further taxonomic and evolutionary studies of Amorphophallus.

A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the ER exit sites (ERESs) and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12 and Sec24, and participates in coat protein II (COPII) vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/ phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER, and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

Phylogenomic analysis and dynamic evolution of chloroplast genomes of Clematis nannophylla.

Clematis nannophylla is a perennial shrub of Clematis with ecological, ornamental, and medicinal value, distributed in the arid and semi-arid areas of northwest China. This study successfully determined the chloroplast (cp) genome of C. nannophylla, reconstructing a phylogenetic tree of Clematis. This cp genome is 159,801 bp in length and has a typical tetrad structure, including a large single-copy, a small single-copy, and a pair of reverse repeats (IRa and IRb). It contains 133 unique genes, including 89 protein-coding, 36 tRNA, and 8 rRNA genes. Additionally, 66 simple repeat sequences, 50 dispersed repeats, and 24 tandem repeats were found; many of the dispersed and tandem repeats were between 20-30 bp and 10-20 bp, respectively, and the abundant repeats were located in the large single copy region. The cp genome was relatively conserved, especially in the IR region, where no inversion or rearrangement was observed, further revealing that the coding regions were more conserved than the noncoding regions. Phylogenetic analysis showed that C. nannophylla is more closely related to C. fruticosa and C. songorica. Our analysis provides reference data for molecular marker development, phylogenetic analysis, population studies, and cp genome processes to better utilise C. nannophylla.

Characterization of the complete chloroplast genome of Rhodiola sachalinensis and comparative analysis with its congeneric plants.

Rhodiola, belonging to the Crassulaceae family, is a perennial herbaceous plant genus. There are about 90 Rhodiola species worldwide, some of which have been reported to have medicinal properties. Rhodiola sachalinensis is a perennial medicinal herb within this genus and, in the present study, its chloroplast genome was sequenced, assembled, annotated and compared with 24 other Rhodiola species. The results obtained show that the chloroplast genome of R. sachalinensis is 151 595 bp long and has a CG content of 37.7%. The inverted repeats (IR) region of the Rhodiola chloroplast genome is the most conserved region, with the main differences being observed in the ycf1 and ndhF genes at the IRb-small single copy boundary, and rps19 and trnH genes at the IRa-large single copy boundary. Phylogenetic analysis showed that Rhodiola species form two major clades, and species with recorded medicinal properties, clustered together in one branch except for R. dumulosa. Within the genus, R. sachalinensis is most closely related to Rhodiola rosea, although comparative analyses showed that only R. sachalinensis and Rhodiola subopposita contained the psbZ gene, which encodes a highly conserved protein subunit of the Photosystem II core complex. Overall, the present study contributes to the understanding of the chloroplast genome of Rhodiola species, and provides a theoretical basis for the study of their genetic diversity and possible use as medicinal plants.

Biased gene introgression and adaptation in the face of chloroplast capture in Aquilegia amurensis.

Chloroplast capture, a phenomenon that can occur through interspecific hybridization and introgression, is frequently invoked to explain cytonuclear discordance in plants. However, relatively few studies have documented the mechanisms of cytonuclear coevolution and its potential for driving species differentiation and possible functional differences in the context of chloroplast capture. To address this crucial question, we chose the Aquilegia genus, which is known for having minimal sterility among species, and inferred that A. amurensis captured the plastome of A. parviflora based on cytonuclear discordance and gene flow between the two species. We focused on the introgression region and its differentiation from corresponding regions in closely related species, especially its composition in a chloroplast capture scenario. We found that nuclear genes encoding cytonuclear enzyme complexes (CECs; i.e., organelle-targeted genes) of chloroplast donor species were selectively retained and displaced the original CEC genes in chloroplast-receiving species due to cytonuclear interactions during introgression. Notably, the intrinsic correlation of CEC introgression was a greater degree of evolutionary distance for these CECs between A. amurensis and A. parviflora. Terpene synthase activity genes (GO: 0010333) were overrepresented among the introgressed genes, and more than 30% of these genes were CEC genes. These findings support our observations that floral terpene release pattern is similar between A. amurensis and A. parviflora compared with A. japonica. Our study clarifies the mechanisms of cytonuclear coevolution, species differentiation and functional differences in the context of chloroplast capture and highlights the potential role of chloroplast capture in adaptation.

Respective role of plastoglobules and lipid droplets in leaf neutral lipid accumulation during senescence and nitrogen deprivation.

Upon abiotic stress or senescence, the size and/or abundancy of plastid-localized plastoglobules and cytosolic lipid droplets, both compartments devoted to neutral lipid storage, increase in leaves. Meanwhile, plant lipid metabolism is also perturbed, notably with the degradation of thylakoidal monogalactosyldiacylglycerol (MGDG) and the accumulation of neutral lipids. Although these mechanisms are probably linked, they have never been jointly studied, and the respective roles of plastoglobules and lipid droplets in the plant response to stress are totally unknown. To address this question, we determined and compared the glycerolipid composition of both lipid droplets and plastoglobules, followed their formation in response to nitrogen starvation and studied the kinetics of lipid metabolism in Arabidopsis leaves. Our results demonstrated that plastoglobules preferentially store phytyl-esters, while triacylglycerols (TAGs) and steryl-esters accumulated within lipid droplets. Thanks to a pulse chase labeling approach and lipid analyses of fatty acid desaturase 2 (fad2) mutant, we showed that MGDG-derived C18:3 fatty acids were exported to lipid droplets, while MGDG-derived C16:3 fatty acids were stored within plastoglobules. The export of lipids from plastids to lipid droplets was likely facilitated by the physical contact occurring between both organelles, as demonstrated by our electron tomography study. The accumulation of lipid droplets and neutral lipids was transient, suggesting that stress-induced TAGs were remobilized during the plant recovery phase by a mechanism that remains to be explored.

PPR proteins in plants: roles, mechanisms, and prospects for rice research.

Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants, with over 300 members in various species. Nearly all PPR proteins are nuclear-encoded and targeted to the chloroplast and mitochondria, modulating organellar gene expression by participating in RNA metabolism, including mRNA stability, RNA editing, RNA splicing, and translation initiation. Organelle RNA metabolism significantly influences chloroplast and mitochondria functions, impacting plant photosynthesis, respiration, and environmental responses. Over the past decades, PPR proteins have emerged as a research focus in molecular biology due to their diverse roles throughout plant life. This review summarizes recent progress in understanding the roles and molecular mechanisms of PPR proteins, emphasizing their functions in fertility, abiotic and biotic stress, grain quality, and chloroplast development in rice. Furthermore, we discuss prospects for PPR family research in rice, aiming to provide a theoretical foundation for future investigations and applications.

The complete chloroplast genome of Marupa (Simarouba amara Aubl., Simaroubaceae).

Marupa (Simarouba amara Aublet 1775) is a tropical tree of the family Simaroubaceae. It is commonly used for its wood in the Amazonian forest, and it is an important species for restoring degraded environments. Yet, very little genetic resources are available to study this plant. In this paper, we sequenced for the first time the complete chloroplast genome of Marupa, using Oxford Nanopore long-read technology. The genome is 159,838 bp, includes 131 genes in total and presents a classic quadripartite structure. Its length and structure are similar to those of sister species of the Simaroubaceae family. A maximum likelihood phylogeny of the order Sapindale reveals that Simarouba amara is well positioned in its family. This complete plastome is a first step towards a better analysis of Marupa future evolution.

The role of chloroplast SRP54 domains and its C-terminal tail region in post- and cotranslational protein transport in vivo.

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the posttranslational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the cotranslational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for posttranslational transport, while a ribosome-associated pool coordinates its cotranslational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis thaliana cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in posttranslational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear- as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.

Plastid genome evolution in leafless members of the orchid subfamily Orchidoideae, with a focus on Degranvillea dermaptera.

PREMISE: Leafless, heterotrophic plants are prime examples of organismal modification, the genomic consequences of which have received considerable interest. In particular, plastid genomes (plastomes) are being sequenced at a high rate, allowing continual refinement of conceptual models of reductive evolution in heterotrophs. However, numerous sampling gaps exist, hindering the ability to conduct comprehensive phylogenomic analyses in these plants. METHODS: Using floral tissue from an herbarium specimen, we sequenced and analyzed the plastome of Degranvillea dermaptera, a rarely collected, leafless orchid species from South America about which little is known, including its phylogenetic affinities. RESULTS: The plastome is the most reduced of those sequenced among the orchid subfamily Orchidoideae. In Degranvillea, it has lost the majority of genes found in leafy autotrophic species, is structurally rearranged, and has similar gene content to the most reduced plastomes among the orchids. We found strong evidence for the placement of Degranvillea within the subtribe Spiranthinae using models that explicitly account for heterotachy, or lineage-specific evolutionary rate variation over time. We further found evidence of relaxed selection on several genes and of correlations among substitution rates and several other "traits" of the plastome among leafless members of orchid subfamily Orchidoideae. CONCLUSIONS: Our findings advance knowledge on the phylogenetic relationships and paths of plastid genome evolution among the orchids, which have experienced more independent transitions to heterotrophy than any other plant family. This study demonstrates the importance of herbarium collections in comparative genomics of poorly known species of conservation concern.

Context-dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

Evolution and Functional Differentiation of the C-terminal Motifs of FtsZs during Plant Evolution.

Filamentous temperature-sensitive Z (FtsZ) is a tubulin-like GTPase that is highly conserved in bacteria and plants. It polymerizes into a ring at the division site of bacteria and chloroplasts and serves as the scaffold protein of the division complex. While a single FtsZ is present in bacteria and cyanobacteria, there are two subfamilies, FtsZ1 and FtsZ2 in the green lineage, and FtsZA and FtsZB in red algae. In Arabidopsis thaliana, the C-terminal motifs of AtFtsZ1 (Z1C) and AtFtsZ2-1 (Z2C) display distinct functions in the regulation of chloroplast division. Z1C exhibits weak membrane-binding activity, whereas Z2C engages in the interaction with the membrane protein AtARC6. Here, we provide evidence revealing the distinct traits of the C-terminal motifs of FtsZ1 and FtsZ2 throughout the plant evolutionary process. In a range of plant species, the C-terminal motifs of FtsZ1 exhibit diverse membrane-binding properties critical for regulating chloroplast division. In chlorophytes, the C-terminal motifs of FtsZ1 and FtsZ2 exhibit both membrane-binding and protein interaction functions, which are similar to those of cyanobacterial FtsZ and red algal FtsZA. During the transition from algae to land plants, the functions of the C-terminal motifs of FtsZ1 and FtsZ2 exhibit differentiation. FtsZ1 lost the function of interacting with ARC6 in land plants, and the membrane-binding activity of FtsZ2 was lost in ferns. Our findings reveal the functional differentiation of the C-terminal motifs of FtsZs during plant evolution, which is critical for chloroplast division.

Impacts of Drought on Photosynthesis in Major Food Crops and the Related Mechanisms of Plant Responses to Drought.

Drought stress is one of the most critical threats to crop productivity and global food security. This review addresses the multiple effects of drought on the process of photosynthesis in major food crops. Affecting both light-dependent and light-independent reactions, drought leads to severe damage to photosystems and blocks the electron transport chain. Plants face a CO2 shortage provoked by stomatal closure, which triggers photorespiration; not only does it reduce carbon fixation efficiency, but it also causes lower overall photosynthetic output. Drought-induced oxidative stress generates reactive oxygen species (ROS) that damage cellular structures, including chloroplasts, further impairing photosynthetic productivity. Plants have evolved a variety of adaptive strategies to alleviate these effects. Non-photochemical quenching (NPQ) mechanisms help dissipate excess light energy as heat, protecting the photosynthetic apparatus under drought conditions. Alternative electron pathways, such as cyclical electron transmission and chloroplast respiration, maintain energy balance and prevent over-reduction of the electron transport chain. Hormones, especially abscisic acid (ABA), ethylene, and cytokinin, modulate stomatal conductance, chlorophyll content, and osmotic adjustment, further increasing the tolerance to drought. Structural adjustments, such as leaf reordering and altered root architecture, also strengthen tolerance. Understanding these complex interactions and adaptive strategies is essential for developing drought-resistant crop varieties and ensuring agricultural sustainability.

Plastomes of Nothofagus reflect a shared biogeographic history in Patagonia.

Plastomes are used in phylogenetic reconstructions because of their relatively conserved nature. Nonetheless, some limitations arise, particularly at lower taxonomic levels due to reduced interspecific polymorphisms and frequent hybridization events that result in unsolved phylogenies including polytomies and reticulate evolutionary patterns. Next-generation sequencing technologies allow access to genomic data and strongly supported phylogenies, yet biased topologies may be obtained due to insufficient taxon sampling. We analyse the hypothesis that intraspecific plastome diversity reflects biogeographic history and hybridization cycles among taxa. We generated twelve new plastome sequences covering distinct latitudinal locations of all species of subgenus Nothofagus from North Patagonia. Chloroplast genomes were assembled, annotated, and searched for simple sequence repeats (SSRs). Phylogenetic reconstructions included species and sampled locations. The six Nothofagus species analysed were of similar size and structure; only N. obliqua of subgenus Lophozonia, used as outgroup, presented slight differences in size. We detected a variable number of SSRs in distinct species and locations. Phylogenetic analyses of plastomes confirmed that subgenus Nothofagus organizes into two monophyletic clades each consisting of individuals of different species. We detected a geographic structure within subgenus Nothofagus and found evidence of local chloroplast sharing due to past hybridization, followed by adaptive introgression and ecological divergence. These contributions enrich the comprehension of transversal evolutionary mechanisms such as chloroplast capture and its implications for phylogenetic and phylogenomic analyses.

Mass spectrometry imaging of Arabidopsis thaliana with in vivo D2O labeling.

The commonly used analytical tools for metabolomics cannot directly probe metabolic activities or distinguish metabolite differences between cells and suborgans in multicellular organisms. These issues can be addressed by in-vivo isotope labeling and mass spectrometry imaging (MSI), respectively, but the combination of the two, a newly emerging technology we call MSIi, has been rarely applied to plant systems. In this study, we explored MSIi of Arabidopsis thaliana with D2O labeling to study and visualize D-labeling in three classes of lipids: arabidopsides, chloroplast lipids, and epicuticular wax. Similar to other stress responses, D2O-induced stress increased arabidopsides in an hour, but it was relatively minor for matured plants and reverted to the normal level in a few hours. The D-labeling isotopologue patterns of arabidopsides matched with those of galactolipid precursors, supporting the currently accepted biosynthesis mechanism. Matrix-assisted laser desorption/ionization (MALDI)-MSI was used to visualize the spatiotemporal distribution of deuterated chloroplast lipids, pheophytin a, MGDGs, and DGDGs, after growing day-after-sowing (DAS) 28 plants in D2O condition for 3-12 days. There was a gradual change of deuteration amount along the leaf tissues and with a longer labeling time, which was attributed to slow respiration leading to low D2O concentration in the tissues. Finally, deuterium incorporation in epicuticular wax was visualized on the surfaces of the stem and flower. The conversion efficiency of newly synthesized C30 aldehyde to C29 ketone was very low in the lower stem but very high at the top of the stem near the flower or on the flower carpel. This study successfully demonstrated that MSIi can unveil spatiotemporal metabolic activities in various tissues of A. thaliana.

Identification of a highly drought-resistant pp7l hda6 mutant.

Plants have developed efficient strategies to counteract drought stress, including stomata closure, significant changes in nuclear gene expression, and epigenetic mechanisms. Previously, we identified Arabidopsis thaliana PROTEIN PHOSPHATASE7-LIKE (PP7L) as an extrachloroplastic protein that promotes chloroplast development. In addition, it was shown that PP7L is involved in high light and salt tolerance. Here, we demonstrate that the pp7l mutant can withstand prolonged periods of drought stress. Interestingly, despite impaired growth under standard growth conditions, photosynthetic efficiency recovers in pp7l mutant plants experiencing drought conditions. To assess the (post)transcriptional changes occurring in the pp7l mutant under different durations of drought exposure, we used an RNA-sequencing technique that allows the simultaneous detection of organellar and nuclear transcripts. Compared with the previously reported drought-responsive changes in the wild type, the drought-responsive changes in organellar and nuclear transcripts detected in the pp7l mutant were negligible. Our analysis of the data generated in this study and review and analysis of previous literature motivated us to create a pp7l hda6 (histone deacetylase 6) mutant, which exhibits remarkable drought resistance. Notably, the growth penalty associated with pp7l was alleviated in the double mutant, ruling out a dwarf effect on the drought-tolerant trait of this genotype. Future studies may consider that multiple loci and factors are involved in stress resistance and explore combinations of these factors to create even more resilient plants.