GBF family member PfGBF3 and NAC family member PfNAC2 regulate rosmarinic acid biosynthesis under high light.

Rosmarinic acid (RA) is an important medicinal metabolite and a potent food antioxidant. We discovered that exposure to high light intensifies the accumulation of RA in the leaves of perilla (Perilla frutescens (L.) Britt). However, the molecular mechanism underlying RA synthesis in response to high light stress remains poorly understood. To address this knowledge gap, we conducted a comprehensive analysis employing transcriptomic sequencing, transcriptional activation, and genetic transformation techniques. High light treatment for 1 and 48 h resulted in the upregulation of 592 and 1,060 genes, respectively. Among these genes, three structural genes and 93 transcription factors exhibited co-expression. Notably, NAC family member PfNAC2, GBF family member PfGBF3, and cinnamate-4-hydroxylase gene PfC4H demonstrated significant co-expression and upregulation under high light stress. Transcriptional activation analysis revealed that PfGBF3 binds to and activates the PfNAC2 promoter. Additionally, both PfNAC2 and PfGBF3 bind to the PfC4H promoter, thereby positively regulating PfC4H expression. Transient overexpression of PfNAC2, PfGBF3, and PfC4H, as well as stable transgenic expression of PfNAC2, led to a substantial increase in RA accumulation in perilla. Consequently, PfGBF3 acts as a photosensitive factor that positively regulates PfNAC2 and PfC4H, while PfNAC2 also regulates PfC4H to promote RA accumulation under high light stress. The elucidation of the regulatory mechanism governing RA accumulation in perilla under high light conditions provides a foundation for developing a high-yield RA system and a model to understand light-induced metabolic accumulation.

HHL1 and SOQ1 synergistically regulate nonphotochemical quenching in Arabidopsis.

Nonphotochemical quenching (NPQ) is an important photoprotective mechanism that quickly dissipates excess light energy as heat. NPQ can be induced in a few seconds to several hours; most studies of this process have focused on the rapid induction of NPQ. Recently, a new, slowly induced form of NPQ, called qH, was found during the discovery of the quenching inhibitor suppressor of quenching 1 (SOQ1). However, the specific mechanism of qH remains unclear. Here, we found that hypersensitive to high light 1 (HHL1)-a damage repair factor of photosystem II-interacts with SOQ1. The enhanced NPQ phenotype of the hhl1 mutant is similar to that of the soq1 mutant, which is not related to energy-dependent quenching or other known NPQ components. Furthermore, the hhl1 soq1 double mutant showed higher NPQ than the single mutants, but its pigment content and composition were similar to those of the wildtype. Overexpressing HHL1 decreased NPQ in hhl1 to below wildtype levels, whereas NPQ in hhl1 plants overexpressing SOQ1 was lower than that in hhl1 but higher than that in the wildtype. Moreover, we found that HHL1 promotes the SOQ1-mediated inhibition of plastidial lipoprotein through its von Willebrand factor type A domain. We propose that HHL1 and SOQ1 synergistically regulate NPQ.

High light triggers flavonoid and polysaccharide synthesis through DoHY5-dependent signaling in Dendrobium officinale.

Dendrobium officinale is edible and has medicinal and ornamental functions. Polysaccharides and flavonoids, including anthocyanins, are important components of D. officinale that largely determine the nutritional quality and consumer appeal. There is a need to study the molecular mechanisms regulating anthocyanin and polysaccharide biosynthesis to enhance D. officinale quality and its market value. Here, we report that high light (HL) induced the accumulation of polysaccharides, particularly mannose, as well as anthocyanin accumulation, resulting in red stems. Metabolome and transcriptome analyses revealed that most of the flavonoids showed large changes in abundance, and flavonoid and polysaccharide biosynthesis was significantly activated under HL treatment. Interestingly, DoHY5 expression was also highly induced. Biochemical analyses demonstrated that DoHY5 directly binds to the promoters of DoF3H1 (involved in anthocyanin biosynthesis), DoGMPP2, and DoPMT28 (involved in polysaccharide biosynthesis) to activate their expression, thereby promoting anthocyanin and polysaccharide accumulation in D. officinale stems. DoHY5 silencing decreased flavonoid- and polysaccharide-related gene expression and reduced anthocyanin and polysaccharide accumulation, whereas DoHY5 overexpression had the opposite effects. Notably, naturally occurring red-stemmed D. officinale plants similarly have high levels of anthocyanin and polysaccharide accumulation and biosynthesis gene expression. Our results reveal a previously undiscovered role of DoHY5 in co-regulating anthocyanin and polysaccharide biosynthesis under HL conditions, improving our understanding of the mechanisms regulating stem color and determining nutritional quality in D. officinale. Collectively, our results propose a robust and simple strategy for significantly increasing anthocyanin and polysaccharide levels and subsequently improving the nutritional quality of D. officinale.

SNHG18 inhibits bladder cancer cell proliferation by increasing p21 transcription through destabilizing c-Myc protein.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been confirmed to play important roles in various cancers including bladder cancer (BC). The precise expression pattern of lncRNA small nucleolar RNA host gene 18 (SNHG18) in BC and its mechanisms of action have not been fully explored. MATERIALS AND METHODS: The expression of SNHG18 was evaluated by RT-qPCR in bladder cancer clinical samples and human bladder cancer cell lines, and stable cell lines overexpressing SNHG18 were constructed. The effect of SNHG18 on the proliferation of bladder cancer cells was detected by soft agar colony formation test, ATP activity test and subcutaneous tumorigenesis model in nude mice. The specific mechanism of SNHG18 inhibition of bladder cancer proliferation was studied by flow cytometry, western blotting, dual luciferase reporter gene assay and protein degradation assay. RESULTS: We found that SNHG18 is significantly downregulated in BC tissues and cell lines. Kaplan-Meier analysis showed that SNHG18 expression is positively correlated with survival in BC patients. Ectopic overexpression of SNHG18 significantly inhibited the proliferation of BC cells in vitro and in vivo. Further mechanistic investigations demonstrated that SNHG18 inhibited c-Myc expression by modulating the ubiquitination-proteasome pathway and that c-Myc is the critical transcription factor that mediates SNHG18 inhibition of BC growth by directly binding to the p21 promoter, which was attributed with significant p21 accumulation. CONCLUSIONS: SNHG18 promotes the transcription and expression of p21 by inhibiting c-Myc expression, leading to G0-G1 arrest and inhibiting the proliferation of bladder cancer cells. These findings highlight a novel cell cycle regulatory mechanism involving the SNHG18/c-Myc/p21 pathway in BC pathogenesis and could potentially lead to new lncRNA-based diagnostics and/or therapeutics for BC.

LINC00955 suppresses colorectal cancer growth by acting as a molecular scaffold of TRIM25 and Sp1 to Inhibit DNMT3B-mediated methylation of the PHIP promoter.

BACKGROUND: Long non-coding RNAs play an important role in the development of colorectal cancer (CRC), while many CRC-related lncRNAs have not yet been identified. METHODS: The relationship between the expression of LINC00955 (Long Intergenic Non-protein Coding RNA 955) and the prognosis of colorectal cancer patients was analyzed using the sequencing results of the TCGA database. LINC00955 expression levels were measured using qRT-PCR. The anti-proliferative activity of LINC00955 was evaluated using CRC cell lines in vitro and xenograft models in nude mice in vivo. The interaction of TRIM25-Sp1-DNMT3B-PHIP-CDK2 was analyzed by western blotting, protein degradation experiment, luciferase, RNA-IP, RNA pull-down assays and immunohistochemically analysis. The biological roles of LINC00955, tripartite motif containing 25 (TRIM25), Sp1 transcription factor (Sp1), DNA methyltransferase 3 beta (DNMT3B), pleckstrin homology domain interacting protein (PHIP), cyclin dependent kinase 2 (CDK2) in colorectal cancer cells were analyzed using ATP assays, Soft agar experiments and EdU assays. RESULTS: The present study showed that LINC00955 is downregulated in CRC tissues, and such downregulation is associated with poor prognosis of CRC patients. We found that LINC00955 can inhibit CRC cell growth both in vitro and in vivo. Evaluation of its mechanism of action showed that LINC00955 acts as a scaffold molecule that directly promotes the binding of TRIM25 to Sp1, and promotes ubiquitination and degradation of Sp1, thereby attenuating transcription and expression of DNMT3B. DNMT3B inhibition results in hypomethylation of the PHIP promoter, in turn increasing PHIP transcription and promoting ubiquitination and degradation of CDK2, ultimately leading to G0/G1 growth arrest and inhibition of CRC cell growth. CONCLUSIONS: These findings indicate that downregulation of LINC00955 in CRC cells promotes tumor growth through the TRIM25/Sp1/DNMT3B/PHIP/CDK2 regulatory axis, suggesting that LINC00955 may be a potential target for the therapy of CRC.

Quantitative proteomics reveals redox-based functional regulation of photosynthesis under fluctuating light in plants.

Photosynthesis involves a series of redox reactions and is the major source of reactive oxygen species in plant cells. Fluctuating light (FL) levels, which occur commonly in natural environments, affect photosynthesis; however, little is known about the specific effects of FL on the redox regulation of photosynthesis. Here, we performed global quantitative mapping of the Arabidopsis thaliana cysteine thiol redox proteome under constant light and FL conditions. We identified 8857 redox-switched thiols in 4350 proteins, and 1501 proteins that are differentially modified depending on light conditions. Notably, proteins related to photosynthesis, especially photosystem I (PSI), are operational thiol-switching hotspots. Exposure of wild-type A. thaliana to FL resulted in decreased PSI abundance, stability, and activity. Interestingly, in response to PSI photodamage, more of the PSI assembly factor PSA3 dynamically switches to the reduced state. Furthermore, the Cys199 and Cys200 sites in PSA3 are necessary for its full function. Moreover, thioredoxin m (Trx m) proteins play roles in redox switching of PSA3, and are required for PSI activity and photosynthesis. This study thus reveals a mechanism for redox-based regulation of PSI under FL, and provides insight into the dynamic acclimation of photosynthesis in a changing environment.

Light Signaling-Dependent Regulation of PSII Biogenesis and Functional Maintenance.

Light is a key environmental cue regulating photomorphogenesis and photosynthesis in plants. However, the molecular mechanisms underlying the interaction between light signaling pathways and photosystem function are unknown. Here, we show that various monochromatic wavelengths of light cooperate to regulate PSII function in Arabidopsis (Arabidopsis thaliana). The photoreceptors cryptochromes and phytochromes modulate the expression of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173), which is required for PSII biogenesis by regulating PSII core protein D1 synthesis mediated by the transcription factor ELONGATED HYPOCOTYL5 (HY5). HY5 directly binds to the ACGT-containing element ACE motif and G-box cis-element present in the HCF173 promoter and regulates its activity. PSII activity was decreased significantly in hy5 mutants under various monochromatic wavelengths of light. Interestingly, we demonstrate that HY5 also directly regulates the expression of the genes associated with PSII assembly and repair, including ALBINO3, HCF136, HYPERSENSITIVE TO HIGH LIGHT1, etc., which is required for the functional maintenance of PSII under photodamaging conditions. Moreover, deficiency of HY5 broadly decreases the accumulation of other photosystem proteins besides PSII proteins. Thus, our study reveals an important role of light signaling in both biogenesis and functional regulation of the photosystem and provides insight into the link between light signaling and photosynthesis in land plants.

LOW PHOTOSYNTHETIC EFFICIENCY 1 is required for light-regulated photosystem II biogenesis in Arabidopsis.

Photosystem II (PSII), a multisubunit protein complex of the photosynthetic electron transport chain, functions as a water-plastoquinone oxidoreductase, which is vital to the initiation of photosynthesis and electron transport. Although the structure, composition, and function of PSII are well understood, the mechanism of PSII biogenesis remains largely elusive. Here, we identified a nuclear-encoded pentatricopeptide repeat (PPR) protein LOW PHOTOSYNTHETIC EFFICIENCY 1 (LPE1; encoded by At3g46610) in Arabidopsis, which plays a crucial role in PSII biogenesis. LPE1 is exclusively targeted to chloroplasts and directly binds to the 5' UTR of psbA mRNA which encodes the PSII reaction center protein D1. The loss of LPE1 results in less efficient loading of ribosome on the psbA mRNA and great synthesis defects in D1 protein. We further found that LPE1 interacts with a known regulator of psbA mRNA translation HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) and facilitates the association of HCF173 with psbA mRNA. More interestingly, our results indicate that LPE1 associates with psbA mRNA in a light-dependent manner through a redox-based mechanism. This study enhances our understanding of the mechanism of light-regulated D1 synthesis, providing important insight into PSII biogenesis and the functional maintenance of efficient photosynthesis in higher plants.

Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

Because bladder cancer (BC) is one of the most common malignant cancers of the urinary system, identification of BC cell growth-associated effectors is of great significance. Cyclin-dependent kinase (CDK)6 is a member of the CDK family of cell cycle-related proteins and plays an important role in cancer cell growth. This is borne out by the fact that a CDK6 inhibitor had been approved to treat several types of cancers. Nevertheless, underlying molecular mechanisms concerning how to regulate CDK6 expression in BC remains unclear. In the present study, it was observed that miR-934 was much higher in human BCs and human BC cell lines as well. The results also revealed that miR-934 inhibition dramatically decreased human BC cell monolayer growth in vitro and xenograft tumor growth in vivo; the outcomes were accompanied by CDK6 protein down-regulation and G0-G1 cell cycle arrest. Moreover, overexpression of CDK6 reversed the inhibition of BC cell growth induced by miR-934. Further studies showed that miR-934 binds to a 3'-UTR of ubiquitin-conjugating enzyme 2N (ube2n) mRNA, down-regulated UBE2N protein expression; this, in turn, attenuated CDK6 protein degradation and led to CDK6 protein accumulation as well as the promotion of BC tumor growth. Collectively, this study not only establishes a novel regulatory axis of miR-934/UBE2N of CDK6 but also provides data suggesting that miR-934 and UBE2N may be potentially promising targets for therapeutic strategies against BC.-Yan, H., Ren, S., Lin, Q., Yu, Y., Chen, C., Hua, X., Jin, H., Lu, Y., Zhang, H., Xie, Q., Huang, C., Huang, H. Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

miR-3687 Overexpression Promotes Bladder Cancer Cell Growth by Inhibiting the Negative Effect of FOXP1 on Cyclin E2 Transcription.

Cyclin E2, a member of the cyclin family, is a key cell cycle-related protein. This protein plays essential roles in cancer progression, and, as such, an inhibitor of cyclin E2 has been approved to treat several types of cancers. Even so, mechanisms underlying how to regulate cyclin E2 expression in cancer remain largely unknown. In the current study, miR-3687 was upregulated in clinical bladder cancer (BC) tumor tissues, The Cancer Genome Atlas (TCGA) database, and human BC cell lines. Inhibition of miR-3687 expression significantly reduced human BC cell proliferation in vitro and tumor growth in vivo, which coincided with the induction of G0/G1 cell cycle arrest and downregulation of cyclin E2 protein expression. Interestingly, overexpression of cyclin E2 reversed the inhibition of BC proliferation induced by miR-3687. Mechanistic studies suggested that miR-3687 binds to the 3' UTR of foxp1 mRNA, downregulates FOXP1 protein expression, and in turn promotes the transcription of cyclin E2, thereby promoting the growth of BC cells. Collectively, the current study not only establishes a novel regulatory axis of miR-3687/FOXP1 regarding regulation of cyclin E2 expression in BC cells, but also provides strong suggestive evidence that miR-3687 and FOXP1 may be promising targets in therapeutic strategies for human BC.

Plastid ribosomal protein LPE2 is involved in photosynthesis and the response to C/N balance in Arabidopsis thaliana.

The balance between cellular carbon (C) and nitrogen (N) must be tightly coordinated to sustain optimal growth and development in plants. In chloroplasts, photosynthesis converts inorganic C to organic C, which is important for maintenance of C content in plant cells. However, little is known about the role of chloroplasts in C/N balance. Here, we identified a nuclear-encoded protein LOW PHOTOSYNTHETIC EFFICIENCY2 (LPE2) that it is required for photosynthesis and C/N balance in Arabidopsis. LPE2 is specifically localized in the chloroplast. Both loss-of-function mutants, lpe2-1 and lpe2-2, showed lower photosynthetic activity, characterized by slower electron transport and lower PSII quantum yield than the wild type. Notably, LPE2 is predicted to encode the plastid ribosomal protein S21 (RPS21). Deficiency of LPE2 significantly perturbed the thylakoid membrane composition and plastid protein accumulation, although the transcription of plastid genes is not affected obviously. More interestingly, transcriptome analysis indicated that the loss of LPE2 altered the expression of C and N response related genes in nucleus, which is confirmed by quantitative real-time-polymerase chain reaction. Moreover, deficiency of LPE2 suppressed the response of C/N balance in physiological level. Taken together, our findings suggest that LPE2 plays dual roles in photosynthesis and the response to C/N balance.

NFκB2 p52 stabilizes rhogdiß mRNA by inhibiting AUF1 protein degradation via a miR-145/Sp1/USP8-dependent axis.

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiß mRNA stability and expression. Positively regulation of rhogdiß mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiß mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIß axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.

Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin (ISO) on human bladder cancer cells.

There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.

p63α protein up-regulates heat shock protein 70 expression via E2F1 transcription factor 1, promoting Wasf3/Wave3/MMP9 signaling and bladder cancer invasion.

Bladder cancer (BC) is the sixth most common cancer in the United States and is the number one cause of death among patients with urinary system malignancies. This makes the identification of invasive regulator(s)/effector(s) as the potential therapeutic targets for managing BC a high priority. p63 is a member of the p53 family of tumor suppressor genes/proteins, plays a role in the differentiation of epithelial tissues, and is believed to function as a tumor suppressor. However, it remains unclear whether and how p63 functions in BC cell invasion after tumorigenesis. Here, we show that p63α protein levels were much higher in mouse high-invasive BC tissues than in normal tissues. Our results also revealed that p63α is crucial for heat shock protein 70 (Hsp70) expression and subsequently increases the ability of BC invasion. Mechanistic experiments demonstrated that p63α can transcriptionally up-regulate Hsp70 expression, thereby promoting BC cell invasion via the Hsp70/Wasf3/Wave3/MMP-9 axis. We further show that E2F transcription factor 1 (E2F1) mediates p63α overexpression-induced Hsp70 transcription. We also found that p63α overexpression activates E2F1 transcription, which appears to be stimulated by p63α together with E2F1. Collectively, our results demonstrate that p63α is a positive regulator of BC cell invasion after tumorigenesis, providing significant insights into the biological function of p63α in BC and supporting the notion that p63α might be a potential target for invasive BC therapy.

XIAP overexpression promotes bladder cancer invasion in vitro and lung metastasis in vivo via enhancing nucleolin-mediated Rho-GDIß mRNA stability.

Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway.

The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis.

Iron (Fe) is an indispensable micronutrient for plant growth and development. The regulation of Fe homeostasis in plants is complex and involves a number of transcription factors. Here, we demonstrate that a basic helix-loop-helix (bHLH) transcription factor, bHLH104, belonging to the IVc subgroup of bHLH family, acts as a key component positively regulating Fe deficiency responses. Knockout of bHLH104 in Arabidopsis thaliana greatly reduced tolerance to Fe deficiency, whereas overexpression of bHLH104 had the opposite effect and led to accumulation of excess Fe in soil-grown conditions. The activation of Fe deficiency-inducible genes was substantially suppressed by loss of bHLH104. Further investigation showed that bHLH104 interacted with another IVc subgroup bHLH protein, IAA-LEUCINE RESISTANT3 (ILR3), which also plays an important role in Fe homeostasis. Moreover, bHLH104 and ILR3 could bind directly to the promoters of Ib subgroup bHLH genes and POPEYE (PYE) functioning in the regulation of Fe deficiency responses. Interestingly, genetic analysis showed that loss of bHLH104 could decrease the tolerance to Fe deficiency conferred by the lesion of BRUTUS, which encodes an E3 ligase and interacts with bHLH104. Collectively, our data support that bHLH104 and ILR3 play pivotal roles in the regulation of Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE expression.

M-type thioredoxins are involved in the xanthophyll cycle and proton motive force to alter NPQ under low-light conditions in Arabidopsis.

KEY MESSAGE: M-type thioredoxins are required to regulate zeaxanthin epoxidase activity and to maintain the steady-state level of the proton motive force, thereby influencing NPQ properties under low-light conditions in Arabidopsis. Non-photochemical quenching (NPQ) helps protect photosynthetic organisms from photooxidative damage via the non-radiative dissipation of energy as heat. Energy-dependent quenching (qE) is a major constituent of NPQ. However, the mechanism underlying the regulation of qE is not well understood. In this study, we demonstrate that the m-type thioredoxins TRX-m1, TRX-m2, and TRX-m4 (TRX-ms) interact with the xanthophyll cycle enzyme zeaxanthin epoxidase (ZE) and are required for maintaining the redox-dependent stabilization of ZE by regulating its intermolecular disulfide bridges. Reduced ZE activity and accumulated zeaxanthin levels were observed under TRX-ms deficiency. Furthermore, concurrent deficiency of TRX-ms resulted in a significant increase in proton motive force (pmf) and acidification of the thylakoid lumen under low irradiance, perhaps due to the significantly reduced ATP synthase activity under TRX-ms deficiency. The increased pmf, combined with acidification of the thylakoid lumen and the accumulation of zeaxanthin, ultimately contribute to the elevated stable qE in VIGS-TRX-m2m4/m1 plants under low-light conditions. Taken together, these results indicate that TRX-ms are involved in regulating NPQ-dependent photoprotection in Arabidopsis.

Optimization of Light-Harvesting Pigment Improves Photosynthetic Efficiency.

Maximizing light capture by light-harvesting pigment optimization represents an attractive but challenging strategy to improve photosynthetic efficiency. Here, we report that loss of a previously uncharacterized gene, HIGH PHOTOSYNTHETIC EFFICIENCY1 (HPE1), optimizes light-harvesting pigments, leading to improved photosynthetic efficiency and biomass production. Arabidopsis (Arabidopsis thaliana) hpe1 mutants show faster electron transport and increased contents of carbohydrates. HPE1 encodes a chloroplast protein containing an RNA recognition motif that directly associates with and regulates the splicing of target RNAs of plastid genes. HPE1 also interacts with other plastid RNA-splicing factors, including CAF1 and OTP51, which share common targets with HPE1. Deficiency of HPE1 alters the expression of nucleus-encoded chlorophyll-related genes, probably through plastid-to-nucleus signaling, causing decreased total content of chlorophyll (a+b) in a limited range but increased chlorophyll a/b ratio. Interestingly, this adjustment of light-harvesting pigment reduces antenna size, improves light capture, decreases energy loss, mitigates photodamage, and enhances photosynthetic quantum yield during photosynthesis. Our findings suggest a novel strategy to optimize light-harvesting pigments that improves photosynthetic efficiency and biomass production in higher plants.