Takehito Inaba

Chloroplast development is primarily regulated by transcription factors, including those belonging to the GOLDEN2-LIKE (GLK) family, that alter the expression of genes encoding chloroplast proteins. However, the mechanisms by which multiple transcription factors coordinate to optimize chloroplast development remain unclear. In this study, we identified the single B-BOX (BBX) CONSTANS-LIKE (COL) proteins COL6 (also known as BBX14), COL7 (BBX16), COL8 (BBX17), and COL16 (BBX15) as novel regulators of chloroplast development in Arabidopsis thaliana. Notably, the col6 col16 (col-d) double mutant exhibited a chlorophyll-excess phenotype, which was further enhanced in the quadruple col6 col7 col8 col16 (col-q) mutant. Introduction of col-d and col-q mutations in the glk1 glk2 (glk-d) mutant partially restored the pale-green phenotype of the original glk-d mutant. COL proteins negatively and redundantly regulated a class of genes encoding chloroplast proteins, acting directly on their promoters. Moreover, COL6 and COL7 expression exhibited a diurnal cycle, with their expression patterns contrasting with those of GLK1 and GLK2, which are positive regulators of chloroplast development. Furthermore, GLK expression was markedly higher in the col-q mutants than in wild-type plants. Co-immunoprecipitation assays revealed that COL proteins interacted with the GARP DNA-binding domain in the N-terminus of GLK1, likely affecting its function. Moreover, COL proteins associate with each other to form homomeric and heteromeric complexes, suggesting functional redundancies at the protein level. In conclusion, these results suggest that the multilayered regulatory mechanisms of single BBX COL and GLK proteins optimize chloroplast development in Arabidopsis thaliana in fluctuating environments.

Sugar transporter SWEETs play diverse roles in plants. To assess the role of SWEETs in floral thermogenesis, we analyzed Symplocarpus renifolius SWEET2, SWEET4, and SWEET6, whose mRNA expression increased from the prethermogenic to thermogenic stages in RNA-seq data. Yeast complementation using hexose transport-deficient strains showed that SrSWEET4 and SrSWEET6 transport both glucose and fructose; SrSWEET4 also transports galactose. SrSWEET2 did not complement hexose transport deficiency in the yeast. Intracellular localization of GFP-tagged proteins in S. renifolius leaf protoplasts revealed that SrSWEET4 and SrSWEET6 localize to the plasma membrane, whereas SrSWEET2 may localize to the tonoplast. In qPCR, SrSWEET4 and SrSWEET6 exhibited prominent organ-specific expression in the spadix, particularly in the stamen, whereas SrSWEET2 was not differentially expressed at any stage or organ. In situ localization of SrSWEET6 mRNA was detected in developing pollen. These results suggest that SrSWEET4 and SrSWEET6 are involved in stamen development via hexose transport during thermogenesis.

Plants use CO2 to produce carbohydrates via photosynthesis. A number of studies have focused on the high CO2 response of plants, whereas little is known about the low CO2 response of plants. Considering that phytohormones regulate responses to various environmental stresses, we hypothesized that they might also regulate the low CO2 response. In this study, we show that Arabidopsis plants adjust the low CO2 response by inducing salicylic acid (SA) and abscisic acid (ABA). Phytohormone analysis revealed a dramatic increase in SA under low CO2 conditions. Furthermore, the increase in SA was completely abolished in the SA induction-deficient2 mutant, indicating that SA is primarily synthesized via isochorismate synthase under low CO2 conditions. The level of ABA also increased under low CO2 conditions. Consistent with these observations, the expression of genes involved in SA and ABA accumulation was altered in response to low CO2 concentrations. Gene expression analysis indicated that the expression of SA- and ABA-responsive genes increased under low CO2 conditions, although the SA response of Pathogenesis-Related genes was compromised. Under low CO2 conditions, it became evident that the expression of programmed cell death-associated genes was altered, and that of photosynthesis-associated genes was suppressed. The responses of these genes were further manifested when plants accumulated high levels of SA. Taken together, we suggest that plants increase their levels of SA and ABA in response to low CO2 environments. Furthermore, the increase in SA appears to be associated with the suppression of non-essential genes and the alteration of cell death-associated genes.

The identification of chemical compounds that affect intracellular processes has greatly contributed to the understanding of developmental regulation in plants. In this protocol, we describe a method for identifying chemical compounds that affect cold-regulated gene expression in Arabidopsis thaliana. Specifically, we generated Arabidopsis plants harboring a COLD-REGULATED 15A (COR15A) promoter::luciferase (COR15Apro::LUC) construct and grew them in a submerged liquid culture. Using a single true leaf excised from COR15Apro::LUC plants and 96-well culture plates, we performed high-throughput screening of chemical compounds that inhibit cold-induction of COR15Apro::LUC. Luciferase activity was detected using a microplate reader and a chemiluminescence imaging device. This protocol can be easily used for the identification of chemical compounds that regulate other processes, being versatile with respect to equipment.

Plants have evolved various mechanisms to adapt to the ever‐changing external environment. Autophagy is one such mechanism and has been suggested to play a key role in responding to and adapting to abiotic stresses in plants. However, the role of autophagy in adaptation to cold and freezing stresses remains to be characterized in detail. Here, we investigated the role of autophagy in the low‐temperature response of Arabidopsis using atg mutants. Both the atg5‐1 and atg10‐1 mutants exhibited normal freezing tolerance, regardless of cold acclimation. A comparison of fresh weights indicated that the difference in growth between the wild‐type and atg plants under cold conditions was rather small compared with that under normal conditions. Analysis of COLD‐REGULATED gene expression showed no significant differences between the atg mutants and wild type. Treatment with 3‐methyladenine, an inhibitor of autophagy, did not impair the induction of COR15Apro::LUC expression upon exposure to low temperature. Evaluation of autophagic activity using transgenic plants expressing RBCS‐mRFP demonstrated that autophagy was rarely induced by cold exposure, even in the dark. Taken together, these data suggest that autophagy is suppressed by low temperatures and is dispensable for cold acclimation and freezing tolerance in Arabidopsis.

In floral thermogenesis, sugars play an important role not only as energy providers but also as growth and development facilitators. Yet, the mechanisms underlying sugar translocation and transport in thermogenic plants remain to be studied. Asian skunk cabbage (Symplocarpus renifolius) is a species that can produce durable and intense heat in its reproductive organ, the spadix. Significant morphological and developmental changes in the stamen are well‐characterized in this plant. In this study, we focused on the sugar transporters (STPs), SrSTP1 and SrSTP14, whose genes were identified by RNA‐seq as the upregulated STPs during thermogenesis. Real‐time PCR confirmed that mRNA expression of both STP genes was increased from the pre‐thermogenic to the thermogenic stage in the spadix, where it is predominantly expressed in the stamen. SrSTP1 and SrSTP14 complemented the growth defects of a hexose transporter‐deficient yeast strain, EBY4000, on media containing 0.02, 0.2, and 2% (w/v) glucose and galactose. Using a recently developed transient expression system in skunk cabbage leaf protoplasts, we revealed that SrSTP1 and SrSTP14‐GFP fusion proteins were mainly localized to the plasma membrane. To dig further into the functional analysis of SrSTPs, tissue‐specific localization of SrSTPs was investigated by in situ hybridization. Using probes for SrSTP14, mRNA expression was observed in the microspores within the developing anther at the thermogenic female stage. These results indicate that SrSTP1 and SrSTP14 transport hexoses (e.g., glucose and galactose) at the plasma membrane and suggest that SrSTP14 may play a role in pollen development through the uptake of hexoses into pollen precursor cells.

The identification of chemical compounds that affect intracellular processes has greatly contributed to our understanding of plant growth and development. In most cases, these compounds have been identified in germinated seedlings. However, chemical screening using mature plants would benefit and advance our understanding of environmental responses. In this study, we developed a high-throughput screening method using single leaves of mature plants to identify small molecules that affect cold-regulated gene expression. A single excised leaf of Arabidopsis (Arabidopsis thaliana) grown in submerged cultures responded to low temperatures in terms of COLD-REGULATED (COR) gene expression. We used transgenic Arabidopsis harboring a COLD-REGULATED 15A (COR15A) promoter::luciferase (COR15Apro::LUC) construct to screen natural compounds that affect the cold induction of COR15Apro::LUC. This approach allowed us to identify derivatives of 1,4-naphthoquinone as specific inhibitors of COR gene expression. Moreover, 1,4-naphthoquinones appeared to inhibit the rapid induction of upstream C-REPEAT BINDING FACTOR (CBF) transcription factors upon exposure to low temperature, suggesting that 1,4-naphthoquinones alter upstream signaling processes. Our study offers a chemical screening scheme for identifying compounds that affect environmental responses in mature plants. This type of analysis is likely to reveal an unprecedented link between certain compounds and plant environmental responses.

Key message Floral thermogenesis is an important reproductive strategy for attracting pollinators. We developed essential biological tools for studying floral thermogenesis using two species of thermogenic aroids, Symplocarpus renifolius and Alocasia odora. Abstract Aroids contain many species with intense heat-producing abilities in their inflorescences. Several genes have been proposed to be involved in thermogenesis of these species, but biological tools for gene functional analyses are lacking. In this study, we aimed to develop a protoplast-based transient expression (PTE) system for the study of thermogenic aroids. Initially, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense as well as durable heat. In this plant, leaf protoplasts were isolated from potted and shoot tip-cultured plants with high efficiency (ca. 1.0 × 10⁵/g fresh weight), and more than half of these protoplasts were successfully transfected. Using this PTE system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). These three GFP-fused proteins were localized in MitoTracker-stained mitochondria in leaf protoplasts, although the green fluorescent particles in protoplasts expressing SrUCPA-GFP were significantly enlarged. Finally, to assess whether the PTE system established in the leaves of S. renifolius is applicable for floral tissues of thermogenic aroids, inflorescences of S. renifolius and another thermogenic aroid (Alocasia odora) were used. Although protoplasts were successfully isolated from several tissues of the inflorescences, PTE systems worked well only for the protoplasts isolated from the female parts (slightly thermogenic or nonthermogenic) of A. odora inflorescences. Our developed system has a potential to be widely used in inflorescences as well as leaves in thermogenic aroids and therefore may be a useful biological tool for investigating floral thermogenesis.

In the originally published version of this manuscript, the “In a Nutshell” summary was mistakenly omitted by the journal’s editorial team from both the uncorrected and corrected proof. It has been now been restored to the article.

Plastids are involved in phytohormone metabolism as well as photosynthesis. However, the mechanism by which plastid retrograde signals and phytohormones cooperatively regulate plastid biogenesis remains elusive. Here, we investigated the effects of an inhibitor and a mutation that generate biogenic plastid signals on phytohormones and vice versa. Inhibition of plastid biogenesis by norflurazon (NF) treatment and the plastid protein import2 (ppi2) mutation caused a decrease in salicylic acid (SA) and jasmonic acid (JA). This effect can be attributed in part to the altered expression of genes involved in the biosynthesis and the metabolism of SA and JA. However, SA-dependent induction of the PATHOGENESIS-RELATED1 gene was virtually unaffected in NF-treated plants and the ppi2 mutant. Instead, the level of chlorophyll in these plants was partially restored by the exogenous application of SA. Consistent with this observation, the levels of some photosynthesis-associated proteins increased in the ppi2 and NF-treated plants in response to SA treatment. This regulation in true leaves seems to occur at the posttranscriptional level since SA treatment did not induce the expression of photosynthesis-associated genes. In salicylic acid induction deficient 2 and lesions simulating disease resistance 1 mutants, endogenous SA regulates the accumulation of photosynthesis-associated proteins through transcriptional and posttranscriptional mechanisms. These data indicate that SA acts antagonistically to the inhibition of plastid biogenesis by promoting the accumulation of photosynthesis-associated proteins in Arabidopsis, suggesting a possible link between SA and biogenic plastid signaling.