Rubisco Research Articles

Proteomic and Biological Analyses Reveal the Effect on Growth under Flooding Stress of Chickpea Irradiated with Millimeter Waves.

Chickpea cultivated on marginal lands in arid and semiarid tropics is one of the food legumes, and its growth is reduced by flooding stress. Millimeter-wave irradiation has influences on organisms, and it improves the growth of plants such as soybean. To reveal the dynamic effects of millimeter-wave irradiation on chickpea under flooding, gel- and label-free proteomic analysis was conducted. Millimeter-wave irradiation improved chickpea growth and its tolerance to flooding stress. According to functional categorization, oppositely changed proteins were correlated with photosynthesis, fermentation, and protein degradation. Immunoblot analysis confirmed that RuBisCO activase and large subunits decreased in leaves under flooding; however, they are recovered in irradiated chickpea even if it was in this condition. The activity and accumulation of alcohol dehydrogenase increased in roots under flooding; however, this followed the same pattern. Cell death was significantly increased and decreased by flooding on unirradiated and irradiated chickpeas, respectively. These findings suggest that irradiation with millimeter waves on chickpea seeds improves the recovery of plant growth through regulation of photosynthesis in leaves and fermentation in roots. Furthermore, millimeter-wave irradiation might promote chickpea tolerance under flooding via the regulation of cell death.

Comprehensive Proteome and Lysine Acetylome Analysis Reveals the Widespread Involvement of Acetylation in Cold Resistance of Pepper (Capsicum annuum L.).

Pepper is a typical warmth-loving vegetable that lacks a cold acclimation mechanism and is sensitive to cold stress. Lysine acetylation plays an important role in diverse cellular processes, but limited knowledge is available regarding acetylation modifications in the resistance of pepper plants to cold stress. In this study, the proteome and acetylome of two pepper varieties with different levels of cold resistance were investigated by subjecting them to cold treatments of varying durations followed by recovery periods. In total, 6,213 proteins and 4,574 lysine acetylation sites were identified, and this resulted in the discovery of 3,008 differentially expressed proteins and 768 differentially expressed acetylated proteins. A total of 1,988 proteins were identified in both the proteome and acetylome, and the functional differences in these co-identified proteins were elucidated through GO enrichment. KEGG analysis showed that 397 identified acetylated proteins were involved in 93 different metabolic pathways. The dynamic changes in the acetylated proteins in photosynthesis and the “carbon fixation in the photosynthetic organisms” pathway in pepper under low-temperature stress were further analyzed. It was found that acetylation of the PsbO and PsbR proteins in photosystem II and the PsaN protein in photosystem I could regulate the response of pepper leaves to cold stress. The acetylation levels of key carbon assimilation enzymes, such as ribulose bisphosphate carboxylase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, and triosephosphate isomerase decreased, leading to decreases in carbon assimilation capacity and photosynthetic efficiency, reducing the cold tolerance of pepper leaves. This study is the first to identify the acetylome in pepper, and it greatly expands the catalog of lysine acetylation substrates and sites in Solanaceae crops, providing new insights for posttranslational modification studies.

Gerbera POE1 (GhPOE1) is involved in leaf senescence in Arabidopsis

Senescence, the final stage of plant development, is a complex and highly controlled physiological process. Senescence occurs in various tissues and organs, and is especially apparent in leaves. Pollen Ole e 1 (POE1) proteins are known to initiate pollen germination, participate in the response to various stresses, and be involved in root development and seed germination. However, their role in plant senescence is not well defined. Using transcriptome data, we isolated a novel protein from Gerbera hybrida (gerbera) which is an ortholog of AT2G34700 (which encodes a Pollen Ole e 1 domain protein in Arabidopsis thaliana) and named GhPOE1. This protein is localized in the cytoplasm and expresses mainly in disk florets, old leaves, and old roots. We transformed GhPOE1 into wild-type Arabidopsis (Col-0) and obtained homozygous transgenic lines OE2, OE3 and OE5. Overexpression of GhPOE1 in these lines led to yellowing rosette leaves with decreased chlorophyll content and photochemical efficiency. Moreover, senescence-associated genes such as SAG12, SAG13, WRKY53, and SIRK (senescence-induced receptor-like kinase) were significantly up-regulated, and the photosynthesis-associated genes CAB2 (chlorophyll a/b-binding 2) and RBCS (ribulose bisphosphate carboxylase small chain) were down-regulated in the transgenic plants. Our results showed that GhPOE1 is a positive regulator of senescence that can promote leaf senescence in Arabidopsis. GhPOE1 is a new candidate gene for genetic engineering to prolong the vase life of cut gerbera flowers.

Phase Equilibria on the Reciprocal Quaternary System K+, Rb+ // Cl–, and Borate–H2O at T = 323.2 K and p = 94.77 kPa

The isothermal dissolution method was applied for the phase equilibrium experiment of the quaternary system K+, Rb+ // Cl–, and borate–H2O at T = 323.2 K and p = 94.77 kPa. The solubilities and refractive indices (nD) of the system were determined experimentally. The coexisting salts of the quaternary invariant points were identified using the X-ray diffraction (XRD) method. Based on the measured data, the stable phase diagram and the figures of water content and refractive index (nD) vs composition were plotted for this system at 323.2 K. The results show that the system belongs to a complex type with the formation of a solid solution [(K, Rb)Cl]. The stable phase diagram consists of three quaternary invariant points, seven isothermal dissolution curves, and five crystallization regions corresponding to KCl, RbCl, RbB5O6(OH)4·2H2O, K2B4O5(OH)4·2H2O, and [(K, Rb)Cl]. The size of the RbB5O6(OH)4·2H2O crystallization zone is the largest among the coexisting salts in the quaternary system, which means it is the most easily crystallized from the mixed solution containing potassium, rubidium, chloride, and borate.

Shared and tailored common bean transcriptomic responses to combined fusarium wilt and water deficit

Common bean (Phaseolus vulgaris L.), one of the most consumed food legumes worldwide, is threatened by two main constraints that are found frequently together in nature, water deficit (WD) and fusarium wilt (Fop). To understand the shared and unique responses of common bean to Fop and WD, we analyzed the transcriptomic changes and phenotypic responses in two accessions, one resistant and one susceptible to both stresses, exposed to single and combined stresses. Physiological responses (photosynthetic performance and pigments quantification) and disease progression were also assessed. The combined FopWD imposition negatively affected the photosynthetic performance and increased the susceptible accession disease symptoms. The susceptible accession revealed a higher level of transcriptional changes than the resistant one, and WD single stress triggered the highest transcriptional changes. While 89 differentially expressed genes were identified exclusively in combined stresses for the susceptible accession, 35 were identified in the resistant one. These genes belong mainly to “stress”, “signaling”, “cell wall”, “hormone metabolism”, and “secondary metabolism” functional categories. Among the up-regulated genes with higher expression in the resistant accession, the cysteine-rich secretory, antigen 5 and Pr-1 (CAP) superfamily protein, a ribulose bisphosphate carboxylase family protein, and a chitinase A seem promising targets for multiple stress breeding.

Chlorella vulgaris phycoremediation at low Cu+2 contents: Proteomic profiling of microalgal metabolism related to fatty acids and CO2 fixation

The aim of this work was to correlate metabolic changes with copper ions (Cu+2) bioremediation by microalgae C. vulgaris 097 CCMA-UFSCar at low Cu+2 content. The metabolic effects include proteome changes related to fatty acid biosynthesis (value-added product) and carbon fixation (climate change mitigation). Cu+2, even at low concentration, showed a significant negative impact on C. vulgaris growth. The microalgal bioremediation reached 100, 74, 38 and 26% for Cu+2 content at 0.1; 0.3; 0.6 and 0.9 mg L−1, respectively. Regarding proteomics, the numbers of proteins reduced (≈37%) from 581 proteins (control) to 369 proteins (0.9 mg of Cu+2 L−1) compared to control. The microalgal CO2 fixation is strictly related to the Calvin cycle, particularly phase 1, in which ribulose bisphosphate carboxylase large chain (RuBisCO) produces two phosphoglycerate molecules from CO2 and ribulose 1,5-bisphosphate. Then, phosphoglycerate can be metabolically reduced into glucose. When compared to control, the RuBisCO was underexpressed (≈50%). Similar changes in proteomic profiling of metabolism-related to fatty acid biosynthesis was observed. Nevertheless, no protein was found for the cultivation at 0.9 mg of Cu+2 L−1. Thus, the analysis of C. vulgaris proteomic data indicated that even at low concentration, Cu+2 lead to drastic metabolic changes.

First Molecular Record and Distribution of Padina pavonica (Linnaeus) Thivy from the Southern Black Sea

Padina is a brown algal genus widely distributed in warm-temperate and tropical regions. Species-level identification of specimens is often difficult due to intraspecific and interspecific morphological plasticity. This study reveals the results of the phylogenetic relationships of specimens, by analyzing the cytochrome oxidase subunit 3 (Cox3) and RuBisCO large subunit (rbcL) gene sequences. A total of 144 new sequences were analyzed together with other Padina sequences obtained from GenBank. Phylogenetic inference identified one well-resolved clade. All of these analyses showed that this genus has only one species in the Black Sea. This is the first study on the molecular diagnosis of macroalgae in the Black Sea, and it contains the first molecular records of Padina in the Black Sea Region.

Enhancement of Biomass Production in Colony-Forming Green Algae, Botryosphaerella sudetica, Under Mixotrophic Cultivation.

In this study, we characterized the potential of colony-forming green algae, Botryosphaerella sudetica KNUA107, isolated from Ulleung Island, South Korea, as a bioresource and analyzed the effects of mixotrophic cultivation on its bioresource production efficiency. Internal transcribed spacer (ITS) (ITS1, 5.8S, and ITS2), ribulose bisphosphate carboxylase large subunit (rbcL), and elongation factor Tu (tufa) regions were used for molecular identification and phylogenetic analysis. B. sudetica KNUA107 had a strong relationship with the green algae of Botryococcus and Botryosphaerella genera, which are colony-forming species, and was also associated with members of the Neochloris genus. To improve biomass productivity, we tested mixotrophic cultivation conditions using several organic carbon sources. Glucose supplementation stimulated B. sudetica KNUA107 growth and reduced the time needed to reach the stationary phase. In addition, the colony size was 1.5–2.0 times larger with glucose than in photoautotrophic cultures, and settleability improved in proportion to colony size. The total lipid content and biomass productivity were also higher in cultures supplemented with glucose. Among the lipid components, saturated fatty acids and monounsaturated fatty acids had the highest proportion. Our study suggests that B. sudetica KNUA107, which has enhanced efficiency in biomass production and lipid components under mixotrophic cultivation, has high potential as a bioresource.

Retraction: Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii.

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

RNA-Seq Transcriptome Analysis of Potato with Differential Tolerance to Bentazone Herbicide

Potato (Solanum tuberosum), an important food crop worldwide, is threatened by broadleaf weeds. Bentazone is an effective herbicide for controlling weeds; however, as a photosynthesis inhibitor, it can also affect potato plants. Therefore, screening potato seedlings for bentazone resistance and determining the genes involved is essential. Herein, we selected potato varieties with tolerance and sensitivity to bentazone. The photosynthetic rate of sensitive plants was notably affected by bentazone application, whereas the tolerant plants showed a significantly higher photosynthetic rate. We observed 95.7% bentazone degradation within 24 d after application in the tolerant plants. Transcriptome sequencing revealed that the numbers of differentially expressed genes (DEGs) between the tolerant and sensitive potato seedlings were 2703 and 11,024 before and after bentazone application, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the majority of DEGs were enriched in metabolic pathways, biosynthesis of secondary metals, carbon metabolism, glutathione metabolism, and photosynthesis. Polyphenol oxidase (PPO), flavonoid 3′,5′-methyltransferase-like (AOMT3), ribulose bisphosphate carboxylase small chain C (RBCS-C), and chalcone synthase 2 (CHS2) were identified as candidates contributing to bentazone tolerance. These results provide a theoretical basis for selecting potato stress-resistant resources in the future.

Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones.

C4 plants, such as maize, strictly compartmentalize Rubisco to bundle sheath chloroplasts. The molecular basis for the restriction of Rubisco from the more abundant mesophyll chloroplasts is not fully understood. Mesophyll chloroplasts transcribe the Rubisco large subunit gene and, when normally quiescent transcription of the nuclear Rubisco small subunit gene family is overcome by ectopic expression, mesophyll chloroplasts still do not accumulate measurable Rubisco. Here we show that a combination of five ubiquitin promoter-driven nuclear transgenes expressed in maize leads to mesophyll accumulation of assembled Rubisco. These encode the Rubisco large and small subunits, Rubisco assembly factors 1 and 2, and the assembly factor Bundle sheath defective 2. In these plants, Rubisco large subunit accumulates in mesophyll cells, and appears to be assembled into a holoenzyme capable of binding the substrate analog CABP (carboxyarabinitol bisphosphate). Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most probably due to a lack of the substrate ribulose 1,5-bisphosphate and/or Rubisco activase. Overall, this work defines a minimal set of Rubisco assembly factors in planta and may help lead to methods of regulating the C4 pathway.

The First Report of Orobanche laxissima Parasitizing Pomegranate (Punica granatum) in Georgia.

Orobanche laxissima Uhlich & Rätzel (Orobanchaceae) is a polyphagous root parasitic plant distributed in the Caucasus Mountains and Transcaucasia; especially Russia, Georgia, Armenia, Azerbaijan, N.E. Turkey (Piwowarczyk et al. 2019). It infects many wild or sometimes cultivated trees and shrubs, such as Betulaceae, Oleaceae, Fagaceae, Aceraceae, Cornaceae, usually Fraxinus L., Fagus L., Carpinus L. (Piwowarczyk et al. 2019, 2020). Punica granatum L. (Lythraceae), commonly known as pomegranate, is native to the Caucasus, the Himalayas in North Pakistan and Northern India, and is widely cultivated, e.g. in USA and throughout the Mediterranean. Pomegranate is one of the first domesticated fruits and have been used in folk medicine or as a food for centuries. Fruit, seed, leaves, flower, root, or barks extracts have extensive medicinal properties (Shaygannia et al. 2015). Field surveys conducted in south-eastern Georgia in May 2019 revealed extensive infestations of O. laxissima on the roots of P. granatum in one locality in Kakheti Province, near Sighnaghi (41°37,4 N, 45°56,3 E, 480 m elevation), in roadside or hills scrub and cultivated areas of pomegranate. The infection was confirmed by verifying the attachment of the Orobanche to the Punica root. The population of the parasite consisted of at least a ca. thousand shoots, sometimes in one clump was ca. 100 individuals. A single plant of pomegranate was parasitized by few to c.a. a hundred of broomrape plants, and 10 to 20% of the ca. 1 ha location was infested. The main botanical features of the O. laxissima are: i) stem simple, (10-)25-40(-100) cm high, with haustoria; ii) inflorescence usually long to short cylindrical or lax, usually many-flowered; iii) calyx-segments entire or bidentate, rarely with 4 teeth; iv) corolla (16-)22-24(-31) mm long, tubular-bell-shaped; purple, pink, rarely dirty yellow, light brown; v) stigma purple, orange, or yellow (Piwowarczyk et al. 2019, 2020). For molecular analysis, total genomic DNA was extracted from the sample and the plastid gene rbcL (rubisco large subunit) was sequenced and amplified as described in Piwowarczyk et al. (2015). The sequence (1231 bp) was deposited in GenBank (MN384886). BLAST search found that it was most similar to (Query Cover 100%, Per Ident. 100%) O. laxissima (KR260928). To the best of our knowledge, this is the first report of a O. laxissima parasitizing P. granatum. O. laxissima appearing in large numbers on singles pomegranate shrubs can weaken the plants, and reduce flowering and fruiting. In the Caucasus region, O. laxissima was observed in mesophilic forests and shrubs, but our report suggests the possibility of a potential spread to neighboring cultivated areas, especially fruit trees and shrubs. Until now, only one report of pathogenic plants was documented for P. granatum, included Phelipanche aegyptiaca (Pers.) Pomel and O. crenata Forssk. in Israel (Dor et al. 2014).

The relative abundance of wheat Rubisco activase isoforms is post-transcriptionally regulated

Diurnal rhythms and light availability affect transcription–translation feedback loops that regulate the synthesis of photosynthetic proteins. The CO2-fixing enzyme Rubisco is the most abundant protein in the leaves of major crop species and its activity depends on interaction with the molecular chaperone Rubisco activase (Rca). In Triticum aestivum L. (wheat), three Rca isoforms are present that differ in their regulatory properties. Here, we tested the hypothesis that the relative abundance of the redox-sensitive and redox-insensitive Rca isoforms could be differentially regulated throughout light–dark diel cycle in wheat. While TaRca1-β expression was consistently negligible throughout the day, transcript levels of both TaRca2-β and TaRca2-α were higher and increased at the start of the day, with peak levels occurring at the middle of the photoperiod. Abundance of TaRca-β protein was maximal 1.5 h after the peak in TaRca2-β expression, but the abundance of TaRca-α remained constant during the entire photoperiod. The redox-sensitive TaRca-α isoform was less abundant, representing 85% of the redox-insensitive TaRca-β at the transcript level and 12.5% at the protein level. Expression of Rubisco large and small subunit genes did not show a consistent pattern throughout the diel cycle, but the abundance of Rubisco decreased by up to 20% during the dark period in fully expanded wheat leaves. These results, combined with a lack of correlation between transcript and protein abundance for both Rca isoforms and Rubisco throughout the entire diel cycle, suggest that the abundance of these photosynthetic enzymes is post-transcriptionally regulated.

Supplementing Blood Diet With Plant Nectar Enhances Egg Fertility in Stomoxys calcitrans.

Stomoxys calcitrans (stable fly) is a cosmopolitan biting fly of both medical and veterinary importance. Unlike blood-feeding-related behavior of stable fly, its plant feeding, the fitness value, and the S. calcitrans–plant interaction are less understood. Here we show based on two chloroplast DNA genes, ribulose bisphosphate carboxylase large chain (rbcL) and the intergenic spacer gene trnH-psbA, that field-collected male and female stable flies fed on various plant species. We investigated the fitness cost of plant feeding using Parthenium hysterophorus, one of the plant species identified to have been fed on by the field-collected flies. Supplementation of blood feeding with a flowering P. hysterophorus plant as nectar source enhanced egg hatchability significantly as compared to blood alone, showing the fitness value of nectar supplementation. However, nectar supplementation did not affect the number of eggs laid or longevity of S. calcitrans as compared to flies that fed on blood alone. S. calcitrans maintained on sugar alone failed to lay eggs. The various plants stable flies fed on demonstrated chemodiversity with their own signature scent. The behavioral response of S. calcitrans to these signature compounds varied from strong attraction (γ-terpinene) to neutral (linalool oxide and myrcene) to repellency (butanoic acid). Our study demonstrated that stable flies feed on nectar, and plant nectar supplementation of blood feeding enhanced larval emergence. Thus, our result has implication in stable fly reproduction, survival, disease transmission, boosting laboratory colony, and the possibility of using plant-derived odors for mass trapping of stable fly, for instance, using γ-terpinene.

DNA barcoding aids in identification of adulterants of Trillium govanianum Wall. ex D. Don

The correct scientific identification of the plant material is crucial to the safety and efficacy of herbal products. Trillium govanianum Wall. ex D. Don, a pharmaceutically prized medicinal plant species endemic to the Himalaya, has been recently subjected to large-scale extraction in the wild due to increasing market demand. Consequently, adulteration and/or substitution of the traded material of T. govanianum with closely-related plants such as Paris polyphylla Sm. has become a quality control problem for the herbal industry. Therefore the present study aimed to (i) develop a reference DNA barcode of T. govanianum and its potent adulterant P. polyphylla for correct identification, (ii) and check adulteration of market samples of T. govanianum. Reference DNA barcode library of T. govanianum and P. polyphylla was successfully established, using Internal transcribed spacer (ITS), Maturase K (matK), chloroplast intergenic spacer (trnH-psbA), and ribulose-bisphosphate carboxylase (rbcL) regions. The ITS, matK and trnH-psbA were found to be ideal reference barcodes for T. govanianum, while ITS and trnH-psbA were suitable for P. polyphylla. All the trade samples (i.e. dried rhizomes), investigated during the present study, got putatively identified with the respective reference barcodes; and thereby indicating the ITS and trnH-psbA regions as the potential DNA barcodes for the identification of the trade samples/adulterants. The ITS region showed the highest mean intra- and inter-specific distances, which proves its high efficiency in differentiating closely related species. Phylogenetic trees were also constructed following the neighbor-joining (NJ) method, based on ITS, rbcL, and trnH-PsbA regions of Trillium and Paris species, which distinguished Trillium species from those of Paris. The availability of a novel DNA barcode for this important medicinal plant species will be helpful for correct identification of its raw plant material, checking illegal trade, and to regulate its sustainable collection from the natural habitats.

A Transcriptomic Approach to Understanding the Combined Impacts of Supra-Optimal Temperatures and CO2 Revealed Different Responses in the Polyploid Coffea arabica and Its Diploid Progenitor C. canephora.

Understanding the effect of extreme temperatures and elevated air (CO2) is crucial for mitigating the impacts of the coffee industry. In this work, leaf transcriptomic changes were evaluated in the diploid C. canephora and its polyploid C. arabica, grown at 25 °C and at two supra-optimal temperatures (37 °C, 42 °C), under ambient (aCO2) or elevated air CO2 (eCO2). Both species expressed fewer genes as temperature rose, although a high number of differentially expressed genes (DEGs) were observed, especially at 42 °C. An enrichment analysis revealed that the two species reacted differently to the high temperatures but with an overall up-regulation of the photosynthetic machinery until 37 °C. Although eCO2 helped to release stress, 42 °C had a severe impact on both species. A total of 667 photosynthetic and biochemical related-DEGs were altered with high temperatures and eCO2, which may be used as key probe genes in future studies. This was mostly felt in C. arabica, where genes related to ribulose-bisphosphate carboxylase (RuBisCO) activity, chlorophyll a-b binding, and the reaction centres of photosystems I and II were down-regulated, especially under 42°C, regardless of CO2. Transcriptomic changes showed that both species were strongly affected by the highest temperature, although they can endure higher temperatures (37 °C) than previously assumed.

Transcriptome profiling reveals histone deacetylase 1 gene overexpression improves flavonoid, isoflavonoid, and phenylpropanoid metabolism in Arachis hypogaea hairy roots.

BackgroundThe peanut (Arachis hypogaea) is a crop plant of high economic importance, but the epigenetic regulation of its root growth and development has not received sufficient attention. Research on Arabidopsis thaliana has shown that histone deacetylases (HDACs) are involved in cell growth, cell differentiation, and stress response. Few studies have focused on the role of HDACs in the root development of other plants, particularly crop plants. In earlier studies, we found large accumulations of A. hypogaea histone deacetylase 1 (AhHDA1) mRNA in peanut roots. However, we did not explore the role of AhHDA1 in peanut root development.MethodsIn this paper, we investigated the role of the peanut AhHDA1 gene and focused on the effect of altered AhHDA1 expression in hairy roots at both the phenotypic and transcriptional levels. We analyzed the transformation of A. hypogaea hairy roots using Agrobacterium rhizogenes and RNA sequencing to identify differentially expressed genes that were assigned to specific metabolic pathways. Transgenic hairy roots were used as experimental material to analyze the downstream genes expression and histone acetylation levels. To thoroughly understand AhHDA1 function, we also simultaneously screened the AhHDA1-interacting proteins using a yeast two-hybrid system.ResultsAhHDA1-overexpressing hairy roots were growth-retarded after 20 d in vitro cultivation, and they had a greater accumulation of superoxide anions and hydrogen peroxide than the control and RNAi groups. AhHDA1 overexpression in hairy roots accelerated flux through various secondary synthetic metabolic pathways, as well as inhibited the primary metabolism process. AhHDA1 overexpression also caused a significant upregulation of genes encoding the critical enzyme chalcone synthase (Araip.B8TJ0, CHS) in the flavonoid biosynthesis pathway, hydroxyisoflavanone synthase (Araip.0P3RJ) in the isoflavonoid biosynthesis pathway, and caffeoyl-CoA O-methyltransferase (Aradu.M62BY, CCoAOMT) in the phenylpropanoid biosynthesis pathway. In contrast, ferredoxin 1 (Araip.327XS), the polypeptide of the oxygen-evolving complex of photosystem II (Araip.N6ZTJ), and ribulose bisphosphate carboxylase (Aradu.5IY98) in the photosynthetic pathway were significantly downregulated by AhHDA1 overexpression. The expression levels of these genes had a positive correlation with histone acetylation levels.ConclusionOur results revealed that the relationship between altered gene metabolism activities and AhHDA1 overexpression was mainly reflected in flavonoid, isoflavonoid, and phenylpropanoid metabolism. AhHDA1 overexpression retarded the growth of transgenic hairy roots and may be associated with cell metabolism status. Future studies should focus on the function of AhHDA1-interacting proteins and their effect on root development.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Papain-like cysteine proteases are required for the regulation of photosynthetic gene expression and acclimation to high light stress.

Chloroplasts are considered to be devoid of cysteine proteases. Using transgenic Arabidopsis lines expressing the rice cystatin, oryzacystatin I (OC-I), in the chloroplasts (PC lines) or cytosol (CYS lines), we explored the hypothesis that cysteine proteases regulate photosynthesis. The CYS and PC lines flowered later than the wild type (WT) and accumulated more biomass after flowering. In contrast to the PC rosettes, which accumulated more leaf chlorophyll and carotenoid pigments than the WT, the CYS lines had lower amounts of leaf pigments. High-light-dependent decreases in photosynthetic carbon assimilation and the abundance of the Rubisco large subunit protein, the D1 protein, and the phosphorylated form of D1 proteins were attenuated in the CYS lines and reversed in the PC lines relative to the WT. However, the transgenic lines had higher amounts of LHC, rbcs, pasbA, and pasbD transcripts than the WT, and also showed modified chloroplast to nucleus signalling. We conclude that cysteine proteases accelerate the reconfiguration of the chloroplast proteome after flowering and in response to high-light stress. Inhibition of cysteine proteases, such as AtCEP1, slows chloroplast protein degradation and stimulates photosynthetic gene expression and chloroplast to nucleus signalling, enhancing stress tolerance traits.

The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii.

ABSTRACTRibulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of control by epistasy of synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feedback on the initiation of LSU translation.