Leaf hydraulic conductance (Kleaf) facilitates the supply of water, enabling continual CO2 uptake while maintaining plant water status. We hypothesized that bundle-sheath and mesophyll cells play key roles in regulating the radial flow of water out of the xylem by responding to abscisic acid (ABA). Thus, we generated transgenic Arabidopsis thaliana plants that are insensitive to ABA in their bundle sheath (BSabi) and mesophyll (MCabi) cells. We also introduced tissue-specific fluorescent markers to distinguish between cells of the palisade mesophyll, spongy mesophyll, and bundle sheath. Both BSabi and MCabi plants showed greater Kleaf and transpiration under optimal conditions. MCabi plants had larger stomatal apertures, higher stomatal index, and greater vascular diameter and biomass relative to the WT and BSabi plants. In response to xylem-fed ABA, both transgenic and WT plants reduced their Kleaf and transpiration. The membrane osmotic water permeability (Pf) of the WT's spongy mesophyll was higher than that of the WT's palisade mesophyll. While the palisade mesophyll maintained a low Pf in response to high ABA, the spongy mesophyll Pf was reduced. Compared to the WT, BSabi bundle-sheath cells had a higher Pf but MCabi spongy mesophyll had an unexpected lower Pf. These results suggest that tissue-specific regulation of Pf by ABA may be confounded by whole-leaf hydraulics and transpiration. ABA increased the symplastic permeability, but its contribution to Kleaf was negligible. We suggest that the bundle sheath-spongy mesophyll pathway dynamically responds to the fluctuations in water availability, while the palisade mesophyll serves as a hydraulic buffer.

CONSTANS (CO) is a critical regulator of flowering that combines photoperiodic and circadian signals in Arabidopsis (Arabidopsis thaliana). CO is expressed in multiple tissues, including seedling roots and young leaves. However, the roles and underlying mechanisms of CO in modulating physiological processes outside of flowering remain obscure. Here, we show that the expression of CO responds to salinity treatment. CO negatively mediated salinity tolerance under long-day (LD) conditions. Seedlings from co mutants were more tolerant to salinity stress, whereas overexpression of CO resulted in plants with reduced tolerance to salinity stress. Further genetic analyses revealed the negative involvement of GIGANTEA (GI) in salinity tolerance requires a functional CO. Mechanistic analysis demonstrated that CO physically interacts with four critical basic leucine zipper (bZIP) transcription factors; ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTOR1 (ABF1), ABF2, ABF3, and ABF4. Disrupting these ABFs made plants hypersensitive to salinity stress, demonstrating that ABFs enhance salinity tolerance. Moreover, ABF mutations largely rescued the salinity-tolerant phenotype of co mutants. CO suppresses the expression of several salinity-responsive genes and influences the transcriptional regulation function of ABF3. Collectively, our results show that the LD-induced CO works antagonistically with ABFs to modulate salinity responses, thus revealing how CO negatively regulates plant adaptation to salinity stress.

NAC (NAM, ATAF1,2, and CUC2) transcription factors (TFs) play critical roles in controlling plant growth, development, and abiotic stress responses. However, few studies have examined NAC proteins related to drought stress tolerance in rose (Rosa chinensis). Here, we identified a drought and abscisic acid (ABA)-induced NAC TF, RcNAC091, that localizes to the nucleus and has transcriptional activation activity. Virus-induced silencing of RcNAC091 resulted in decreased drought stress tolerance, and RcNAC091 overexpression had the opposite effect. Specifically, ABA mediated RcNAC091-regulated drought tolerance. A transcriptomic comparison showed altered expression of genes involved in ABA signaling and oxidase metabolism in RcNAC091-silenced plants. We further confirmed that RcNAC091 directly targets the promoter of RcWRKY71 in vivo and in vitro. Moreover, RcWRKY71-slienced rose plants were not sensitive to both ABA and drought stress, whereas RcWRKY71-overexpressing plants were hypersensitive to ABA, which resulted in drought-tolerant phenotypes. The expression of ABA biosynthesis- and signaling-related genes was impaired in RcWRKY71-slienced plants, suggesting that RcWRKY71 might facilitate the ABA-dependent pathway. Therefore, our results show that RcWRKY71 is transcriptionally activated by RcNAC091, which positively modulates ABA signaling and drought responses. The results of this study provide insights into the roles of TFs as functional links between RcNAC091 and RcWRKY71 in priming resistance; our findings also have implications for the approaches to enhance the drought resistance of roses.

Transfer RNA (tRNA) can produce smaller RNA fragments called tRNA-derived fragments (tRFs). tRFs play critical roles in multiple cellular programs, although the functional mechanisms of tRFs remain largely unknown in plants. In this study, we examined the phenotype associated with 5' tRF-Ala (tRF-Ala, produced from tRNA-Ala) overexpression and knockdown lines (tDR-Ala-OE and tDR-Ala-kd, respectively) and the mechanisms by which tRF-Ala affects mRNA levels in Arabidopsis (Arabidopsis thaliana). We investigated the candidate proteins associated with tRF-Ala by quantitative proteomics and confirmed the direct interaction between tRF-Ala and the splicing factor SERINE-ARGININE RICH PROTEIN 34 (SR34). A transcriptome sequencing analysis showed that 318 genes among all the genes (786) with substantial alternative splicing (AS) variance in tDR-Ala-OE lines are targets of SR34. tRF-Ala diminished the binding affinity between SR34 and its targets by direct competition for interaction with SR34. These findings reveal the critical roles of tRF-Ala in regulating mRNA levels and splicing.

Many tree species have developed extensive root systems that allow them to survive in arid environments by obtaining water from a large soil volume. These root systems can transport and redistribute soil water during drought by hydraulic redistribution (HR). A recent study revealed the phenomenon of evaporation-driven hydraulic redistribution (EDHR), which is driven by evaporative demand (transpiration). In this study, we confirmed the occurrence of EDHR in Chinese white poplar (Populus tomentosa) through root sap flow measurements. We utilized micro-computed tomography technology to reconstruct the xylem network of woody lateral roots and proposed conceptual models to verify EDHR from a physical perspective. Our results indicated that EDHR is driven by the internal water potential gradient within the plant xylem network, which requires three conditions: high evaporative demand, soil water potential gradient, and special xylem structure of the root junction. The simulations demonstrated that during periods of extreme drought, EDHR could replenish water to dry roots and improve root water potential up to 38.9%-41.6%. This highlights the crucial eco-physiological importance of EDHR in drought tolerance. Our proposed models provide insights into the complex structure of root junctions and their impact on water movement, thus enhancing our understanding of the relationship between xylem structure and plant hydraulics.

Thousands of long intergenic noncoding RNAs (lincRNAs) have been identified in plant genomes. While some lincRNAs have been characterized as important regulators in different biological processes, little is known about the transcriptional regulation for most plant lincRNAs. Through the integration of eight annotation resources, we defined 6,599 high-confidence lincRNA loci in Arabidopsis (Arabidopsis thaliana). For lincRNAs belonging to different evolutionary age categories, we identified major differences in sequence and chromatin features, as well as in the level of conservation and purifying selection acting during evolution. Spatiotemporal gene expression profiles combined with transcription factor (TF) chromatin immunoprecipitation data were used to construct a TF-lincRNA regulatory network containing 2,659 lincRNAs and 15,686 interactions. We found that properties characterizing lincRNA expression, conservation and regulation differ between plants and animals. Experimental validation confirmed the role of three TFs, KANADI 1 (KAN1), MYB DOMAIN PROTEIN 44 (MYB44), and PHYTOCHROME INTERACTING FACTOR 4 (PIF4), as key regulators controlling root-specific lincRNA expression, demonstrating the predictive power of our network. Furthermore, we identified 58 lincRNAs, regulated by these TFs, showing strong root cell-type specific expression or chromatin accessibility, which are linked with GWAS genetic associations related to root system development and growth. The multi-level genome-wide characterization covering chromatin state information, promoter conservation, and ChIP-based TF binding, for all detectable lincRNAs across 769 expression samples, permits rapidly defining the biological context and relevance of Arabidopsis lincRNAs through regulatory networks.

Lotus (Nelumbo spp.) is an important aquatic ornamental genus in the family Nelumbonaceae comprising only two species: N. lutea with yellow flowers and N. nucifera with red or white flowers. The petal color variations between these two species have previously been associated with the potential activities of FLAVONOL SYNTHASE (FLS) and MYB5. However, the underlying genetic mechanisms of flower color divergence within the N. nucifera species remains unclear. Here, quantitative trait locus mapping led to the identification of MYB5, a candidate gene controlling petal color in N. nucifera. Genotyping of 213 natural lotus accessions revealed an 80-kb presence/absence variant (PAV) of the NnMYB5 gene that is associated with petal color variation. Transcriptome analysis, dual-luciferase and yeast one-hybrid assays showed that NnMYB5 could directly activate the anthocyanin transporter gene GLUTATHIONE S-TRANSFERASE2 (NnGST2). Heterologous expression of NnGST2 in Arabidopsis (Arabidopsis thaliana) and its overexpression in lotus petals induced anthocyanin accumulation. Deletion of the 80-kb PAV within NnMYB5 inactivated NnGST2 expression and blocked anthocyanin accumulation in white N. nucifera petals. In contrast, the anthocyanin deficiency of N. lutea occurred due to pseudogenized NlMYB5 alleles. Our results establish a regulatory link between NnMYB5 and NnGST2 in petal anthocyanin accumulation and demonstrate the independent mechanisms controlling flower coloration in Nelumbo.

β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the non-reducing ends of β-D-galactosides and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast one-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical ß-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

Single-cell metabolomics is a powerful tool that can reveal cellular heterogeneity and can elucidate the mechanisms of biological phenomena in detail. It is a promising approach in studying plants, especially when cellular heterogeneity has an impact on different biological processes. In addition, metabolomics, which can be regarded as a detailed phenotypic analysis, is expected to answer previously unanswered questions which will lead to expansion of crop production, increased understanding of resistance to diseases, and in other applications as well. In this review, we will introduce the flow of sample acquisition, and single-cell metabolomics techniques to facilitate the adoption of single-cell metabolomics. Furthermore, the applications of single cell metabolomics will be summarized and reviewed.

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by non-plant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed hierarchical assembly starting with sub-particles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These assembled into semi-crystalline α-glucan superstructures up to 4 μm in length filling most of the yeast cytosol. ISA was not essential for formation of such particles, but their abundance was increased dramatically by ISA presence.

There is a limited understanding of the carbon assimilation capacity of non-foliar green tissues and its impact on yield and seed quality since most photosynthesis research focuses on leaf photosynthesis. In this study, we investigate the photosynthetic efficiency of soybean (Glycine max) pods and seeds in a field setting and evaluate its effect on mature seed weight and composition. We demonstrate that soybean pod and seed photosynthesis contributes 13-14% of the mature seed weight. Carbon assimilation by soybean pod and seed photosynthesis can compensate for 81% of carbon loss through the respiration of the same tissues, and our model predicts that soybean pod and seed photosynthesis contributes up to 9% of the total daily carbon gain of the canopy. Chlorophyll fluorescence shows that the operating efficiency of Photosystem II in immature soybean seeds peaks at the 10-100 mg seed weight stage, while that of immature pods peaks at the 75-100 mg stage. This study provides quantitative information about the efficiency of soybean pod and seed photosynthesis during tissue development and its impact on yield.

Varying light conditions elicit metabolic responses as part of acclimation with changes in ascorbate levels being an important component. Here, we adopted a genome-wide association-based approach to characterize the response in ascorbate levels on high light acclimation in a panel of 315 Arabidopsis (Arabidopsis thaliana) accessions. These studies revealed statistically significant SNPs for total and reduced ascorbate under high light conditions at a locus in chromosome 2. Ascorbate levels under high light and the region upstream and within PAS/LOV PROTEIN (PLP) were strongly associated. Intriguingly, subcellular localization analyses revealed that the PLPA and PLPB splice variants co-localized with VITAMIN C DEFECTIVE2 (VTC2) and VTC5 in both the cytosol and nucleus. Yeast 2-hybrid and Bimolecular fluorescence complementation analyses revealed that PLPA and PLPB interact with VTC2 and that blue light diminishes this interaction. Furthermore, PLPB knockout mutants were characterized by 1.5- to 1.7- fold elevations in their ascorbate levels, whereas knockout mutants of the cry2 cryptochromes displayed 1.2- to 1.3- fold elevations compared to WT. Our results collectively indicate that PLP plays a critical role in the elevation of ascorbate levels, which is a signature response of high light acclimation. The results strongly suggest that this is achieved via release of the inhibitory effect of PLP on VTC2 upon blue light illumination, as the VTC2-PLPB interaction is stronger under darkness. The conditional importance of the cryptochrome receptors under different environmental conditions suggests a complex hierarchy underpinning the environmental control of ascorbate levels. However, the data we present here clearly demonstrate that PLP dominates during high light acclimation.

Brassinosteroids (BRs) play an essential role in promoting skotomorphogenesis, yet the underlying mechanisms remain unknown. Here we report that a plant-specific BLISTER (BLI) protein functions as a positive regulator of both BR signaling and skotomorphogenesis in Arabidopsis (Arabidopsis thaliana). We found that the glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2) interacts with and phosphorylates BLI at four phosphorylation sites (Ser70, Ser146, Thr256 and Ser267) for degradation; in turn, BR inhibits degradation of BLI. Specifically, BLI cooperates with the BRASSINAZOLE RESISTANT1 (BZR1) transcription factor to facilitate the transcriptional activation of BR-responsive genes. Genetic analyses indicated that BLI is essentially required for BZR1-mediated hypocotyl elongation in the dark. Intriguingly, we reveal that BLI and BZR1 orchestrate the transcriptional expression of gibberellin (GA) biosynthetic genes to promote the production of bioactive GAs. Our results demonstrate that BLI acts as an essential regulator of Arabidopsis skotomorphogenesis through promoting BR signaling and GA biosynthesis.

Carbon monoxide (CO) is a recently discovered gasotransmitter. In animals, it has been found that endogenously produced CO participates in the regulation of various metabolic processes. Recent research has indicated that CO, acting as a signaling molecule, plays a crucial regulatory role in plant development and their response to abiotic stress. In this work, we developed a fluorescent probe, named COP (carbonic oxide Probe), for the in-situ imaging of carbon monoxide (CO) in Arabidopsis thaliana plant tissues. The probe was designed by combining malononitrile-naphthalene as the fluorophore and a typical palladium-mediated reaction mechanism. When reacted with the released CO, COP showed an obvious fluorescence enhancement at 575 nm, which could be observed in naked-eye conditions. With a linear range of 0-10 μM, the limit of detection of COP was determined as 0.38 μM. The detection system based on COP indicated several advantages including relatively rapid response within 20 min, steadiness in a wide pH range of 5.0-10.0, high selectivity, and applicative anti-interference. Moreover, with a penetration depth of 30 μm, COP enabled 3D imaging of CO dynamics in plant samples, whether it was caused by agent release, heavy metal stress or inner oxidation. This work provides a fluorescent probe for monitoring CO levels in plant samples, it expands the application field of CO detection technology, assisting researchers in understanding the dynamic changes in plant physiological processes, making it an important tool for studying plant physiology and biological processes.

Domestication is the long and complex process underlying the evolution of crops, in which artificial directional selection transformed wild progenitors into the desired form, affecting genomic variation and leaving traces of selection at targeted loci. However, whether genes controlling important domestication traits follow the same evolutionary pattern expected under the standard selective sweep model remains unclear. With whole-genome re-sequencing of mungbean (Vigna radiata), we investigated this issue by resolving its global demographic history and targeted dissection of the molecular footprints of genes underlying two key traits representing different stages of domestication. Mungbean originated in Asia, and the Southeast Asian wild population migrated to Australia about 50 thousand generations ago (kga). Later in Asia, the cultivated form diverged from the wild progenitor. We identified the gene associated with the pod shattering resistance trait, VrMYB26a, with lower expression across cultivars and reduced polymorphism in the promoter region, reflecting a hard selective sweep. On the other hand, the stem determinacy trait was associated with VrDet1. We found two ancient haplotypes of this gene have lower gene expression and exhibited intermediate frequencies in cultivars, consistent with selection favoring independent haplotypes in a soft selective sweep. In mungbean, contrasting signatures of selection were identified from the detailed dissection of two important domestication traits. The results suggest complex genetic architecture underlying the seemingly simple process of directional artificial selection and highlight the limitations of genome-scan methods relying on hard selective sweeps.

The selective turnover of macromolecules by autophagy provides a critical homeostatic mechanism for recycling cellular constituents and for removing superfluous and damaged organelles, membranes, and proteins. To better understand how autophagy impacts seed maturation and nutrient storage, we studied maize (Zea mays) endosperm in its early and middle developmental stages via an integrated multi-omics approach using mutants impacting the core macroautophagy factor AUTOPHAGY (ATG)-12 required for autophagosome assembly. Surprisingly, the mutant endosperm in these developmental windows accumulated normal amounts of starch and Zein storage proteins. However, the tissue acquired a substantially altered metabolome, especially for compounds related to oxidative stress and sulfur metabolism, including increases in cystine, dehydroascorbate, cys-glutathione disulfide, glucarate and galactarate, and decreases in peroxide and the anti-oxidant glutathione. While changes in the associated transcriptome were mild, the proteome was strongly altered in the atg12 endosperm, especially for increased levels of mitochondrial proteins without a concomitant increase in mRNA abundances. Although fewer mitochondria were seen cytologically, a heightened number appeared dysfunctional based on the accumulation of dilated cristae, consistent with attenuated mitophagy. Collectively, our results confirm that macroautophagy plays a minor role in the accumulation of starch and storage proteins during maize endosperm development, but likely helps protect against oxidative stress and clears unneeded/dysfunctional mitochondria during tissue maturation.

Despite the global importance of species with C4 photosynthesis, there is a lack of consensus regarding C4 performance under fluctuating light. Contrasting hypotheses and experimental evidence suggest that C4 photosynthesis is either less or more efficient in fixing carbon under fluctuating light than the ancestral C3 form. Two main issues have been identified that may underly the lack of consensus: neglect of evolutionary distance between selected C3 and C4 species and use of contrasting fluctuating light treatments. To circumvent these issues, we measured photosynthetic responses to fluctuating light across three independent phylogenetically controlled comparisons between C3 and C4 species from Alloteropsis, Flaveria, and Cleome genera under 21% and 2% O2. Leaves were subjected to repetitive stepwise changes in light intensity (800 and 100 µmol m-2 s-1 PFD) with three contrasting durations: 6, 30 and 300 seconds. These experiments reconciled the opposing results found across previous studies and showed that 1) stimulation of CO2 assimilation in C4 species during the low light phase was both stronger and more sustained than in C3 species; 2) CO2 assimilation patterns during the high light phase could be attributable to species or C4 subtype differences rather than photosynthetic pathway; and 3) the duration of each light step in the fluctuation regime can strongly influence experimental outcomes.

Cold and drought stresses severely limit crop production and can occur simultaneously. Although some transcription factors and hormones have been characterized in plants subjected each stress, the role of metabolites, especially volatiles, in response to cold and drought stress exposure is rarely studied due to lack of suitable models. Here, we established a model for studying the role of volatiles in tea (Camellia sinensis) plants experiencing cold and drought stresses simultaneously. Using this model, we showed that volatiles induced by cold stress promote drought tolerance in tea plants by mediating reactive oxygen species and stomatal conductance. Needle trap micro-extraction combined with GC-MS identified the volatiles involved in the crosstalk and showed that cold-induced (Z)-3-hexenol improved the drought tolerance of tea plants. In addition, silencing CsADH2 (Camellia sinensis alcohol dehydrogenase 2) led to reduced (Z)-3-hexenol production and significantly reduced drought tolerance in response to simultaneous cold and drought stress. Transcriptome and metabolite analyses, together with plant hormones comparison and abscisic acid (ABA) biosynthesis pathway inhibition experiments, further confirmed the roles of ABA in (Z)-3-hexenol-induced drought tolerance of tea plants. (Z)-3-hexenol application and gene silencing results supported the hypothesis that (Z)-3-hexenol plays a role in the integration of cold and drought tolerance by stimulating the dual function glucosyltransferase UGT85A53, thereby altering ABA homeostasis in tea plants. Overall, we present a model for studying the roles of metabolites in plants under multiple stresses and reveal the roles of volatiles in integrating cold and drought stresses in plants.

Parasitic plants are globally prevalent pathogens with important ecological functions but also potentially devastating agricultural consequences. Common to all parasites is the formation of the haustorium which requires parasite organ development and tissue invasion into the host. Both processes involve cell wall modifications. Here, we investigated a role for pectins during haustorium development in the facultative parasitic plant Phtheirospermum japonicum. Using transcriptomics data from infected Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), we identified genes for multiple P. japonicum pectin methylesterases (PMEs) and their inhibitors (PMEIs) whose expression was upregulated by haustoria formation. Changes in PME and PMEI expression were associated with tissue-specific modifications in pectin methylesterification. While de-methylesterified pectins were present in outer haustorial cells, highly methylated pectins were present in inner vascular tissues, including the xylem bridge that connects parasite to host. Specifically blocking xylem bridge formation in the haustoria inhibited several PME and PMEI genes from activating. Similarly, inhibiting PME activity using chemicals or by overexpressing PMEI genes delayed haustoria development. Our results suggest a dynamic and tissue-specific regulation of pectin contributes to haustoria initiation and to the establishment of xylem connections between parasite and host.

Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that despite being highly hypoxic under normal oxygen tension, QC stem cells are vulnerable to hypoxic stress, which causes their degradation with subsequent inhibition of root growth. Under low oxygen, QC stem cells became depleted of starch and soluble sugars and exhibited reliance on glycolytic fermentation with the impairment of the TCA cycle through the depressed activity of several enzymes, including pyruvate dehydrogenase (PDH). This finding suggests that carbohydrate delivery from the shoot might be insufficient to meet the metabolic demand of QC stem cells during stress. Some metabolic changes characteristic of the hypoxic response in mature root cells were not observed in the QC. Hypoxia-responsive genes, such as PYRUVATE DECARBOXYLASE (PDC) and ALCOHOL DEHYDROGENASE (ADH), were not activated in response to hypoxia, despite an increase in ADH activity. Increases in phosphoenolpyruvate (PEP) with little change in steady-state levels of succinate were also atypical responses to low oxygen tensions. Over-expression of PHYTOGLOBIN 1 (ZmPgb1.1) preserved the functionality of the QC stem cells during stress. The QC stem cell preservation was underpinned by extensive metabolic rewiring centered around activation of the TCA cycle and retention of carbohydrate storage products, denoting a more efficient energy production and diminished demand for carbohydrates under conditions where nutrient transport may be limiting. Overall, this study provides an overview of metabolic responses occurring in plant stem cells during oxygen deficiency.

In Chlamydomonas (Chlamydomonas reinhardtii) the VESICLE-INDUCING PROTEINS IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiome can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL1-11). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

The endophytic nitrogen-fixing bacterium A02 belongs to the genus Curtobacterium (Curtobacterium sp.) and is crucial for the nitrogen (N) metabolism of cassava(Manihot esculenta Crantz). We isolated the A02 strain from cassava cultivar SC205 and used the 15N isotope dilution method to study the impacts of A02 on growth and accumulation of N in cassava seedlings. Furthermore, the whole genome was sequenced to determine the N-fixation mechanism of A02. Compared with low N control (T1), inoculation with the A02 strain (T2) showed the highest increase in leaf and root dry weight of cassava seedlings, and 120.3 nmol·(mL·h) was the highest nitrogenase activity recorded in leaves, which were considered the main site for colonization and N-fixation. The genome of A02 was 3,555,568 bp in size and contained a circular chromosome and a plasmid. Comparison with the genomes of other short bacilli revealed that strain A02 showed evolutionary proximity to the endophytic bacterium NS330 (Curtobacterium citreum) isolated from rice (Oryza sativa) in India. The genome of A02 contained 13 nitrogen fixation (nif) genes, including 4 nifB, 1 nifR3, 2 nifH, 1 nifU, 1 nifD, 1 nifK, 1 nifE, 1 nifN, and 1 nifC, and formed a relatively complete N fixation gene cluster 8-kb long that accounted for 0.22% of the whole genome length. The nifHDK of strain A02(Curtobacterium sp.) is identical to the Frankia alignment. Function prediction showed high copy number of the nifB gene was related to the oxygen protection mechanism. Our findings provide exciting information about the bacterial genome in relation to N support for transcriptomic and functional studies for increasing N use efficiency in cassava.