Rubisco Small Subunit Research Articles

Genome-Wide Analysis and Expression Profiling of Soybean RbcS Family in Response to Plant Hormones and Functional Identification of GmRbcS8 in Soybean Mosaic Virus.

Rubisco small subunit (RbcS), a core component with crucial effects on the structure and kinetic properties of the Rubisco enzyme, plays an important role in response to plant growth, development, and various stresses. Although Rbcs genes have been characterized in many plants, their muti-functions in soybeans remain elusive. In this study, a total of 11 GmRbcS genes were identified and subsequently divided into three subgroups based on a phylogenetic relationship. The evolutionary analysis revealed that whole-genome duplication has a profound effect on GmRbcSs. The cis-acting elements responsive to plant hormones, development, and stress-related were widely found in the promoter region. Expression patterns based on the RT-qPCR assay exhibited that GmRbcS genes are expressed in multiple tissues, and notably Glyma.19G046600 (GmRbcS8) exhibited the highest expression level compared to other members, especially in leaves. Moreover, differential expressions of GmRbcS genes were found to be significantly regulated by exogenous plant hormones, demonstrating their potential functions in diverse biology processes. Finally, the function of GmRbcS8 in enhancing soybean resistance to soybean mosaic virus (SMV) was further determined through the virus-induced gene silencing (VIGS) assay. All these findings establish a strong basis for further elucidating the biological functions of RbcS genes in soybeans.

Transgenic expression of Rubisco accumulation factor2 and Rubisco subunits increases photosynthesis and growth in maize.

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown.

Magnaporthe oryzae Effector AvrPik-D Targets Rice Rubisco Small Subunit OsRBCS4 to Suppress Immunity.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae (M. oryzae), is a highly destructive disease that significantly impacts rice yield and quality. During the infection, M. oryzae secretes effector proteins to subvert the host immune response. However, the interaction between the effector protein AvrPik-D and its target proteins in rice, and the mechanism by which AvrPik-D exacerbates disease severity to facilitate infection, remains poorly understood. In this study, we found that the M. oryzae effector AvrPik-D interacts with the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) small subunit OsRBCS4. The overexpression of the OsRBCS4 gene in transgenic rice not only enhances resistance to M. oryzae but also induces more reactive oxygen species following chitin treatment. OsRBCS4 localizes to chloroplasts and co-localizes with AvrPik-D within these organelles. AvrPik-D suppresses the transcriptional expression of OsRBCS4 and inhibits Rubisco activity in rice. In conclusion, our results demonstrate that the M. oryzae effector AvrPik-D targets the Rubisco small subunit OsRBCS4 and inhibits its carboxylase and oxygenase activity, thereby suppressing rice innate immunity to facilitate infection. This provides a novel mechanism for the M. oryzae effector to subvert the host immunity to promote infection.

Rubisco small subunit (RbCS) is co-opted by potyvirids as the scaffold protein in assembling a complex for viral intercellular movement.

Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana-Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.

Insights on the enhancement of chilling tolerance in Rice through over-expression and knock-out studies of OsRBCS3

ABSTRACT Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

Exogenous Salicylic Acid Alleviates Water Deficit Stress by Protecting Photosynthetic System in Maize Seedlings

Water deficit is a significant abiotic stress affecting crop growth and production. While many studies have indicated that salicylic acid (SA) plays a crucial role in mitigating the detrimental effects of environmental stress on plants, its mechanism regulating the photosynthetic adaptability of maize seedlings under water deficit is still unclear. This study aimed to investigate the impact of exogenous SA on maize seedling performance under polyethylene glycol (PEG)-induced water deficit. The results showed that PEG treatment destroyed the integrity of chloroplast and reduced chlorophyll content and photosynthesis rate (Pn), leading to growth retardation of maize seedlings with lower biomass accumulation and leaf relative water content (RWC). Moreover, chlorophyll fluorescence index, including potential photochemical activity (Fv/Fo), maximum Photosystem II (PSII) quantum yield (Fv/Fm), and energy captured by PSII reaction center for electron transfer (Eto/RC), were decreased, but energy dissipated by unit reaction center (DIo/RC) was enhanced in maize seedlings under water deficit. In addition, PEG treatment also significantly declined the activity of Rubisco and Rubisco activase (RCA) in maize seedlings. In contrast, SA treatment enhanced the content of chlorophyll, as well as the transcription level of psbA, and RCA and Rubisco small subunit (rbcS) reduced the damaging effects of PEG treatment by protecting the integrity of chloroplast and repairing the damaged PSII reaction center, thus positively regulating photosynthetic reaction and water-deficit tolerance in maize seedlings. Our data implied that SA played an important regulatory role in plant resistance to water-deficit stress, and the result will further supply the regulatory network of SA-mediated photosynthetic adaptability.

Effect of 1-butyl-3-methylimidazolium tetrafluoroborate on photosynthesis in Arabidopsis thaliana

Ionic liquids (ILs) are a promising alternative to the traditional organic solvents, but their toxic effects cause significant adverse environmental effects and their toxicity assessment have been suggested. The effect of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]) on photosynthesis was studied in Arabidopsis thaliana. Apparent effects on chlorosis and inhibition of photosynthetic capacity were observed. As [Bmim][BF4] concentrations increased, a time-dose effect was also observed. The maximum quantum yield (Fv/Fm), and the actual quantum yield of photosystem II (ΦPSII), both decreased; at the same time, the expression of psbA, a key gene of the photosystem II electron transport, was reduced by [Bmim][BF4] treatment. The synthesis of the large and small subunits of Rubisco were not affected by treatment. On the other hand, Rubisco activase (RCA) was significantly suppressed at the transcriptional level. Our results suggest that photochemistry of PSII as well as Rubisco activation are involved in ILs resistance.

Antihypertensive peptide resources map of ribulose-1,5-bisphosphate carboxylase/oxygenases (RuBisCO) in angiosperms: Revealed by an integrated in silico and in vitro approach

As the most abundant protein on earth, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been considered a promising resource of functional foods. This study aimed to explore the full potential of plant RuBisCO proteins as precursors of antihypertensive peptides on a large scale. In total, 12,766 RuBisCO large subunit and 1,020 RuBisCO small subunit sequences of angiosperms were collected for simulated proteolysis and evaluation of antihypertensive potential, revealing a vast reservoir of antihypertensive peptides. Moreover, RuBisCO-derived novel antihypertensive peptides TTVW, TMW, and VPCL were identified with in vitro IC50 of 12.89 ± 0.82, 23.97 ± 1.02, and 339.12 ± 21.64 μM, respectively. Notably, TTVW and TMW are noncompetitive inhibitors predicted to bound adjacent to the catalytic region of ACE, while VPCL is a competitive inhibitor predicted to bound to the central active site inside ACE. Overall, this work provides a powerful theoretical guidance in developing antihypertensive functional foods utilizing plant RuBisCO.

A synthetic light-inducible photorespiratory bypass enhances photosynthesis to improve rice growth and grain yield

Bioengineering of photorespiratory bypasses is an effective strategy for improving plant productivity by modulating photosynthesis. In previous work, two photorespiratory bypasses, the GOC and GCGT bypasses, increased photosynthetic rates but decreased seed-setting rate in rice (Oryza sativa), probably owing to excess photosynthate accumulation in the stem. To solve this bottleneck, we successfully developed a new synthetic photorespiratory bypass (called the GMA bypass) in rice chloroplasts by introducing Oryza sativa glycolate oxidase 1 (OsGLO1), Cucurbita maxima malate synthase (CmMS), and Oryza sativa ascorbate peroxidase7 (OsAPX7) into the rice genome using a high-efficiency transgene stacking system. Unlike the GOC and GCGT bypass genes driven by constitutive promoters, OsGLO1 in GMA plants was driven by a light-inducible Rubisco small subunit promoter (pRbcS); its expression dynamically changed in response to light, producing a more moderate increase in photosynthate. Photosynthetic rates were significantly increased in GMA plants, and grain yields were significantly improved under greenhouse and field conditions. Transgenic GMA rice showed no reduction in seed-setting rate under either test condition, unlike previous photorespiratory-bypass rice, probably reflecting proper modulation of the photorespiratory bypass. Together, these results imply that appropriate engineering of the GMA bypass can enhance rice growth and grain yield without affecting seed-setting rate.

Manipulation of light spectrum is an effective tool to regulate biochemical traits and gene expression in lettuce under different replacement methods of nutrient solution

The use of light-emitting diode (LED) technology represents a promising approach to improve plant growth and metabolic activities. The aim of this study was to investigate the effect of different light spectra: red (656 nm), blue (450 nm), red/blue (3:1), and white (peak at 449 nm) on biochemical properties, photosynthesis and gene expression in two lettuce cultivars (Lollo Rossa and Lollo Bionda) grown under different methods of nutrient solution replacement in hydroponics. Complete replacement and EC-based replacement of nutrient solution increased content of proline and soluble sugars and activity of antioxidant enzymes (CAT, GPX and SOD) under the red/blue LED and red LED light treatments in both cultivars. In addition, the red/blue and the monochromatic red light increased the soluble protein content and the antioxidant activity in the Lollo Rosa cultivar under the replacement method according to the needs of the plant. An increase in flavonoid content in the EC-based method in the Lollo Rosa variety treated with a combination of red and blue light was also observed. The red/blue light had the greatest induction effect on anthocyanin content, expression of the UFGT, CHS, and Rubisco small subunit genes, and the net photosynthetic rate. Data presented here will directly contribute to the development of nutrient solution and LED spectrum management strategies to significantly improve plant growth and metabolism, while avoiding water and nutrient waste, and environmental pollution.

Increased Biomass and Polyhydroxybutyrate Production by Synechocystis sp. PCC 6803 Overexpressing RuBisCO Genes.

The overexpression of the RuBisCO (rbc) gene has recently become an achievable strategy for increasing cyanobacterial biomass and overcoming the biocompound production restriction. We successfully constructed two rbc-overexpressing Synechocystis sp. PCC 6803 strains (OX), including a strain overexpressing a large subunit of RuBisCO (OXrbcL) and another strain overexpressing all large, chaperone, and small subunits of RuBisCO (OXrbcLXS), resulting in higher and faster growth than wild type under sodium bicarbonate supplementation. This increased biomass of OX strains significantly contributed to the higher polyhydroxybutyrate (PHB) production induced by nutrient-deprived conditions, in particular nitrogen (N) and phosphorus (P). As a result of higher PHB contents in OX strains occurring at days 7 and 9 of nutrient deprivation, this enhancement was apparently made possible by cells preferentially maintaining their internal lipids while accumulating less glycogen. The OXrbcLXS strain, with the highest level of PHB at about 39 %w/dry cell weight (DCW) during 7 days of BG11-NP treatment, contained a lower glycogen level (31.9 %w/DCW) than wild type control (40 %w/DCW). In contrast, the wild type control strain exposed to N- and NP-stresses tended to retain lipid levels and store more glycogen than PHB. In this model, we, for the first time, implemented a RuBisCO-overexpressing cyanobacterial factory for overproducing PHB, destined for biofuel and biomaterial biotechnology.

Activity of Arabidopsis Rubisco small subunit promoter in various tissues of chickpea.

The activity of a green tissue-specific promoter of the Rubisco small subunit gene fromArabidopsis(AraSSU) was studied using transgenic chickpea lines. We generated transgenic chickpea lines expressing anAraSSUpromoter-drivencry2Aagene throughthe Agrobacterium-mediatedtransformation method. Lines withAraSSUexpressed the gene in all green tissues at high levels (> 90ng/mg of fresh weight tissue) compared to lines generated usingCaMV35S(< 10ng/mg FW). We used vertical cross sections of various tissues of homozygous progeny using microtome for immunolocalization. The immunolocalization showed the expression of thecry2Aagene in the green mesophyll cells of the leaves of bothAraSSUand CaMV35 chickpea lines. Moreover, the accumulation ofAraSSU-regulatedCry2Aa protein was also observed in vascular tissues, including enucleate sieve elements and their companion cells. However, no expression was observed in the roots ofAraSSUlines. In the case ofCaMV35lines, the transgene expression was observed in all the tissues. Since our data indicated that theAraSSUpromoter is active in non-green tissues such as vascular bundles. Therefore, we validated this by RT-PCR. We found Cry2Aa RNA transcripts in leaves, stems without epidermis (for vascular tissues), and roots with and without epidermis. Thus,the AraSSUpromoter is active in all above-ground tissues of the chickpea plant.

Transcriptome dynamics provides insights into divergences of the photosynthesis pathway between Saccharum officinarum and Saccharum spontaneum.

Saccharum spontaneum and Saccharum officinarum contributed to the genetic background of modern sugarcane cultivars. Saccharum spontaneum has shown a higher net photosynthetic rate and lower soluble sugar than S. officinarum. Here, we analyzed 198 RNA-sequencing samples to investigate the molecular mechanisms for the divergences of photosynthesis and sugar accumulation between the two Saccharum species. We constructed gene co-expression networks based on differentially expressed genes (DEGs) both for leaf developmental gradients and diurnal rhythm. Our results suggested that the divergence of sugar accumulation may be attributed to the enrichment of major carbohydrate metabolism and the oxidative pentose phosphate pathway. Compared with S. officinarum, S. spontaneum DEGs showed a high enrichment ofphotosynthesis and contained more complex regulation of photosynthesis-related genes. Noticeably, S. spontaneum lacked gene interactions with sulfur assimilation stimulated by photorespiration. In S. spontaneum, core genes related to clock and photorespiration displayed a sensitive regulation by the diurnal rhythm and phase-shift. Small subunit of Rubisco (RBCS) displayed higher expression in the source tissues of S. spontaneum. Additionally, it was more sensitive under a diurnal rhythm, and had more complex gene networks than that in S. officinarum. This indicates that the differential regulation of RBCS Rubisco contributed to photosynthesis capacity divergence in both Saccharum species.

Biophysical models for studying patterns of RuBisCO small subunit evolution.

For much of Earth's history, key enzymes have dominated the conversion of energy into the production of biomass. This history extends hundreds of millions of years into the past, but the available record provides few direct indicators of the emergence and evolution of enzymes that shaped our planet. A good example is Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is responsible for the fixation of CO2 in conjunction with oxygenic photosynthesis and is estimated to be the most abundant enzyme on Earth. RuBisCO form-I is the only form composed of a large and small subunit. The biochemical role that the small subunit plays is unclear, and the circumstances of its coevolution with the large subunit or with drastic changes to the Earth's surface environment such as the concentrations of CO2 and O2 gases are also unknown. Here, we explore how the small subunit-mediated RuBisCO form-I transition by generating a phylogenetic tree of proteins, reconstructing its ancestral sequences, and analyzing the structure as well as function of the ancestral large-small subunit RuBisCO complexes. From ancient and extant structures, we identify the general attributes that define the interaction between the two RuBisCO subunits and inspect how these interactions have evolved with reference to the ancestral large subunit RuBisCO forms and the partition and diffusion of CO2 and O2 at different relative concentrations of the two gasses. Our results are consistent with the possibility that the small subunit was an evolutionary response to the global rise of atmospheric O2 over 2 billion years ago, and that it impacted large subunit CO2 diffusion. Our research highlights how a protein subunit that emerged billions of years ago in response to a change in the planet's atmosphere impacts the function of an essential protein complex.

The Effects of Enhanced Ultraviolet-B Radiation on Leaf Photosynthesis and Submicroscopic Structures in Mangifera indica L. cv. ‘Tainong No 1’

Objective: In the future, the stress of enhanced UV-B radiation on the Earth will first affect the photosynthesis of plants, including mangoes. Therefore, it is necessary to study the effects of enhanced UV-B radiation on the photosynthesis of mangoes. Methods: ‘Tainong No 1’ mango trees in the field were selected as the experimental material and divided into 2 groups: one group was shined under 96 kJ·m−2·d−1 UV-B lamps for artificially simulated treatment of enhanced UV-B radiation, and the other group was shined under sunshine directly as the control (CK). The main photosynthetic physiological indicators were measured with conventional methods, and the expression levels of the genes encoding large and small subunits of the Rubisco enzyme were measured with fluorescent qPCR. The changes in stomatal morphology and chloroplast structure were observed with scanning electron microscopy and transmission electron microscopy. Results: The content of malondialdehyde (MDA) and the relative conductivity in the leaves of the treatment tended to be significantly higher than those of the CK. The net photosynthetic rate (Pn) of the treatment tended to decrease and be lower than that of CK. The dynamics of intercellular CO2 concentration (Ci) of the treatment and CK changed differently from each other but generally tended to decrease, and that of the treatment tended to be significantly higher than that of CK. The stomatal conductance (Gs) of the treatment and CK both generally decreased, and that of the treatment was always significantly lower than that of CK. The contents of chlorophyll a, chlorophyll b and total chlorophyll and the ratio of chlorophyll a/b of the treatment were lower than those of CK, while the carotenoid content showed the opposite trend. The stomata and the surface of leaves of the treatment were sunken and damaged, respectively. The palisade tissue, spongy tissue and upper epidermis thickened more, and the total thickness significantly increased. Meanwhile, the ratio of palisade tissue to spongy tissue decreased. During treatment, the chloroplasts were swollen and shortened, the number of chloroplasts was reduced, the starch grains were degraded, and the grana lamella were distorted, loosely arranged and blurred. The expression of the genes encoding the Rubisco large subunit (rbcL) in the treatment was significantly inhibited, while that encoding the Rubisco small subunit (rbcS) decreased first and increased later. In conclusion, 96 kJ·m−2·d−1 enhanced UV-B radiation treatment caused damage to the leaf cell membrane system. This led to stomatal limitation of photosynthesis by destroying the stomatal structure and nonstomatal limitation of photosynthesis by damaging the submicrostructure of the chloroplasts and downregulating the expression of rbcL. The leaves may resist the photosynthetic damage caused by enhanced UV-B radiation by upregulating rbcS expression as much as possible.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Nicotiana benthamiana Kunitz peptidase inhibitor-like protein involved in chloroplast-to-nucleus regulatory pathway in plant-virus interaction.

Plant viruses use a variety of strategies to infect their host. During infection, viruses cause symptoms of varying severity, which are often associated with altered leaf pigmentation due to structural and functional damage to chloroplasts that are affected by viral proteins. Here we demonstrate that Nicotiana benthamiana Kunitz peptidase inhibitor-like protein (KPILP) gene is induced in response to potato virus X (PVX) infection. Using reverse genetic approach, we have demonstrated that KPILP downregulates expression of LHCB1 and LHCB2 genes of antenna light-harvesting complex proteins, HEMA1 gene encoding glutamyl-tRNA reductase, which participates in tetrapyrrole biosynthesis, and RBCS1A gene encoding RuBisCO small subunit isoform involved in the antiviral immune response. Thus, KPILP is a regulator of chloroplast retrograde signaling system during developing PVX infection. Moreover, KPILP was demonstrated to affect carbon partitioning: reduced glucose levels during PVX infection were associated with KPILP upregulation. Another KPILP function is associated with plasmodesmata permeability control. Its ability to stimulate intercellular transport of reporter 2xGFP molecules indicates that KPILP is a positive plasmodesmata regulator. Moreover, natural KPILP glycosylation is indispensable for manifestation of this function. During PVX infection KPILP increased expression leads to the reduction of plasmodesmata callose deposition. These results could indicate that KPILP affects plasmodesmata permeability via callose-dependent mechanism. Thus, virus entering a cell and starting reproduction triggers KPILP expression, which leads to downregulation of nuclear-encoded chloroplast genes associated with retrograde signaling, reduction in photoassimilates accumulation and increase in intercellular transport, creating favorable conditions for reproduction and spread of viral infection.

Specialized metabolism by trichome-enriched Rubisco and fatty acid synthase components.

Acylsugars, specialized metabolites with defense activities, are secreted by trichomes of many solanaceous plants. Several acylsugar metabolic genes (AMGs) remain unknown. We previously reported multiple candidate AMGs. Here, using multiple approaches, we characterized additional AMGs. First, we identified differentially expressed genes between high- and low-acylsugar-producing F2 plants derived from a cross between cultivated tomato (Solanum lycopersicum) and a wild relative (Solanum pennellii), which produce acylsugars that are ∼1% and ∼20% of leaf dry weight, respectively. Expression levels of many known and candidate AMGs positively correlated with acylsugar amounts in F2 individuals. Next, we identified lycopersicum-pennellii putative orthologs with higher nonsynonymous to synonymous substitutions. These analyses identified four candidate genes, three of which showed enriched expression in stem trichomes compared to underlying tissues (shaved stems). Virus-induced gene silencing confirmed two candidates, Sopen05g009610 [beta-ketoacyl-(acyl-carrier-protein) reductase; fatty acid synthase component] and Sopen07g006810 (Rubisco small subunit), as AMGs. Phylogenetic analysis indicated that Sopen05g009610 is distinct from specialized metabolic cytosolic reductases but closely related to two capsaicinoid biosynthetic reductases, suggesting evolutionary relationship between acylsugar and capsaicinoid biosynthesis. Analysis of publicly available datasets revealed enriched expression of Sopen05g009610 orthologs in trichomes of several acylsugar-producing species. Similarly, orthologs of Sopen07g006810 were identified as solanaceous trichome-enriched members, which form a phylogenetic clade distinct from those of mesophyll-expressed "regular" Rubisco small subunits. Furthermore, δ13C analyses indicated recycling of metabolic CO2 into acylsugars by Sopen07g006810 and showed how trichomes support high levels of specialized metabolite production. These findings have implications for genetic manipulation of trichome-specialized metabolism in solanaceous crops.

Rubisco Small Subunits’ Genome-Wide Identification and Their Function from Gene Expression to Rubisco Activity and Photosynthesis among Peanut Genotypes under Different Nitrogen Levels

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a critical protein for photosynthesis. With small subunits, RBCSs catalyze the fixation of CO2 onto ribulose-1,5-bisphosphate (RuBP). However, the gene characteristics of peanut RBCSs, and the relationship between their expression and peanut growth under nitrogen (N)-deficiency conditions are not yet known. This study targeted the impact of sufficient N (5 mmol/L NO3−) and deficient N (0.1 mmol/L NO3−) on peanut seedlings’ growth, physiology, and molecular aspects. Herein, eight RBCS protein encoding genes were identified in the peanut genome, and their characteristics were described. The leaf areas of Yu Hang Hua 1 (YH1) and An Hua 3 (AH3) were reduced significantly after 10 days while “Pu Ke Hua 18” (PK18) and “Luo Hua 4087” (LH4087) had higher N-deficiency adaptations in the initial N-deficiency stage. The Rubisco carboxylation activities of YH1 and PK18 were significantly increased after 5 days of N-deficiency stress while they decreased under 15 days of N-deficiency conditions. Furthermore, the qRT-PCR results showed that the expressions of AhRBCS 1/5 and AhRBCS2/6 had negative relationships with the morpho-physiological traits of peanut under low-N stress. This study provides the biological information of peanut RBCSs and lays a theoretical foundation for N utilization in crop plants.

Temperature-induced changes in Arabidopsis Rubisco activity and isoform expression.

In many plant species, expression of the nuclear encoded Rubisco small subunit (SSu) varies with environmental changes, but the functional role of any changes in expression remains unclear. In this study, we investigated the impact of differential expression of Rubisco SSu isoforms on carbon assimilation in Arabidopsis. Using plants grown at contrasting temperatures (10 °C and 30 °C), we confirm the previously reported temperature response of the four RbcS genes and extend this to protein expression, finding that warm-grown plants produce Rubisco containing ~65% SSu-B and cold-grown plants produce Rubisco with ~65% SSu-A as a proportion of the total pool of subunits. We find that these changes in isoform concentration are associated with kinetic changes to Rubisco in vitro: warm-grown plants produce a Rubisco having greater CO2 affinity (i.e. higher SC/O and lower KC) but lower kcatCO2 at warm measurement temperatures. Although warm-grown plants produce 38% less Rubisco than cold-grown plants on a leaf area basis, warm-grown plants can maintain similar rates of photosynthesis to cold-grown plants at ambient CO2 and 30 °C, indicating that the carboxylation capacity of warm-grown Rubisco is enhanced at warmer measurement temperatures, and is able to compensate for the lower Rubisco content in warm-grown plants. This association between SSu isoform expression and maintenance of Rubisco activity at high temperature suggests that SSu isoform expression could impact the temperature response of C3 photosynthesis.