Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Translational activation of ribosome-related genes at initial photoreception is dependent on signals derived from both the nucleus and the chloroplasts in Arabidopsis thaliana.

Light is one of the indispensable elements that plants need in order to grow and develop. In particular, it is essential for inducing morphogenesis, such as suppression of hypocotyl elongation and cotyledon expansion, that plants undergo when they first emerge after germination. However, there is a lack of knowledge about the gene expression and, in particular, the translational levels that induce a response upon light exposure. We have investigated the translational expression of nuclear genes in Arabidopsis thaliana seedlings germinated in the dark and then exposed to blue monochromatic light. In this study, ribosome profiling analysis was performed in the blue-light-receptor mutant cry1cry2 and the light-signaling mutant hy5 to understand which signaling pathways are responsible for the changes in gene expression at the translational level after blue-light exposure. The analysis showed that the expression of certain chloroplast- and ribosome-related genes was up-regulated at the translational level in the wild type. However, in both mutants the translational up-regulation of ribosome-related genes was apparently compromised. This suggests that light signaling through photoreceptors and the HY5 transcription factor are responsible for translation of ribosome-related genes. To further understand the effect of photoreception by chloroplasts on nuclear gene expression, chloroplast function was inhibited by adding a photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a carotenoid synthesis inhibitor, norflurazon. The results show that inhibition of chloroplast function did not lead to an increase in the expression of ribosome-related genes at the translational level. These results suggest that signals from both the nucleus and chloroplasts are required to activate translation of ribosome-related genes during blue-light reception.

Functional Differentiation among the Arabidopsis Phosphatidylinositol 4-Phosphate 5-Kinase Genes PIP5K1, PIP5K2 and PIP5K3.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is involved in regulating various cellular processes through the signaling function of its product, phosphatidylinositol (4,5)-bisphosphate. Higher plants encode a large number of PIP5Ks forming distinct clades in their molecular phylogenetic tree. Although biological functions of PIP5K genes have been analyzed intensively in Arabidopsis thaliana, it remains unclear how those functions differ across clades of paralogs. We performed comparative functional analysis of the Arabidopsis genes encoding PIP5K1, PIP5K2 and PIP5K3, of which the first two and the last belong to closely related but distinct clades, to clarify their conserved and/or differentiated functions. Genetic analysis with their single and multiple mutants revealed that PIP5K1 and PIP5K3 have non-overlapping functions, with the former in total plant growth and the latter in root hair elongation, whereas PIP5K2 redundantly functions in both phenomena. This pattern of functional redundancy is explainable in terms of the overlapping pattern of their promoter activities. In transformation rescue experiments, PIP5K3 promoter-directed PIP5K1-YFP completely rescued the short-root-hair phenotype of pip5k3. However, PIP5K3-YFP could substitute for PIP5K1-YFP only partially in rescuing the severe dwarfism of pip5k1pip5k2 when directed by the PIP5K1 promoter. Phylogenetic analysis of angiosperm PIP5Ks revealed that PIP5K3 orthologs have a faster rate of diversification in their amino-acid sequences compared with PIP5K1/2 orthologs after they arose through a eudicot-specific duplication event. These findings suggest that PIP5K3 specialized to promote root hair elongation and lost some of the protein-encoded functions retained by PIP5K1 and PIP5K2, whereas PIP5K1 differentiated from PIP5K2 only in its promoter-directed expression pattern.

CFI 25 Subunit of Cleavage Factor I is Important for Maintaining the Diversity of 3' UTR Lengths in Arabidopsis thaliana (L.) Heynh.

Cleavage and polyadenylation at the 3' end of the pre-mRNA is essential for mRNA function, by regulating its translatability, stability and translocation to the cytoplasm. Cleavage factor I (CFI) is a multi-subunit component of the pre-mRNA 3' end processing machinery in eukaryotes. Here, we report that plant CFI 25 subunit of CFI plays an important role in maintaining the diversity of the 3' ends of mRNA. The genome of Arabidopsis thaliana (L.) Heynh. contained four genes encoding three putative CFI subunits (AtCFI 25, AtCFI 59 and AtCFI 68), orthologous to the mammalian CFI subunits. There were two CFI 25 paralogs (AtCFI 25a and AtCFI 25b) that shared homology with human CFI 25. Two null alleles of AtCFI 25a displayed smaller rosette leaves, longer stigmatic papilla, smaller anther, earlier flowering and lower fertility compared to wild-type plants. Null alleles of AtCFI 25b, as well as, plants ectopically expressing full-length cDNA of AtCFI 25a, displayed no obvious morphological defects. AtCFI 25a was shown to interact with AtCFI 25b, AtCFI 68 and itself, suggesting various forms of CFI in plants. Furthermore, we show that AtCFI 25a function was essential for maintaining proper diversity of the 3' end lengths of transcripts coding for CFI subunits, suggesting a self-regulation of the CFI machinery in plants. AtCFI 25a was also important to maintain 3' ends for other genes to different extent. Collectively, AtCFI 25a, but not AtCFI 25b, seemed to play important roles during Arabidopsis development by maintaining proper diversity of the 3' UTR lengths.

Arabidopsis PLDζ1 and PLDζ2 localize to post-Golgi membrane compartments in a partially overlapping manner.

KEY MESSAGE: Arabidopsis PLDζ1 and PLDζ2 localize to the trans-Golgi network and to compartments including the trans-Golgi network, multi-vesicular bodies, and the tonoplast, respectively, depending on their N-terminal regions containing PX-PH domains. Phospholipase D (PLD) is involved in dynamic cellular processes, including membrane trafficking, cytoskeletal reorganization, and signal transduction for gene expression, through the production of phosphatidic acid in membrane compartments specific to each process. Although PLD plays crucial roles in various plant phenomena, the underlying processes involving PLD for each phenomenon remain largely elusive, partly because the subcellular localization of PLD remains obscure. In this study, we performed comparative subcellular localization analyses of the Arabidopsis thaliana PX-PH-PLDs PLDζ1 and PLDζ2. In mature lateral root cap cells, own promoter-driven fluorescence protein fusions of PLDζ1 localized to the entire trans-Golgi network (TGN) while that of PLDζ2 localized to punctate structures including part of the TGN and multi-vesicular bodies as well as the tonoplast. These localization patterns were reproduced using N-terminal partial proteins, which contain PX-PH domains. An inducibly overexpressed fluorescence protein fusion of the PLDζ2 partial protein first localized to punctate structures, and then accumulated predominantly on the tonoplast. Further domain dissection analysis revealed that the N-terminal moiety preceding the PX-PH domain of PLDζ2 was required for the tonoplast-predominant accumulation. These findings suggest that PLDζ1 and PLDζ2 play partially overlapping but nonetheless distinctive roles in post-Golgi compartments along the membrane trafficking pathway from the TGN to the tonoplast.

Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K7, PIP5K8, and PIP5K9 are redundantly involved in root growth adaptation to osmotic stress.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2 ), a signaling phospholipid critical for various cellular processes in eukaryotes. The Arabidopsis thaliana genome encodes 11 PIP5K genes. Of these, three type B PIP5K genes, PIP5K7, PIP5K8, and PIP5K9, constitute a subgroup highly conserved in land plants, suggesting that they retain a critical function shared by land plants. In this study, we comprehensively investigated the biological functions of the PIP5K7-9 subgroup genes. Reporter gene analyses revealed their preferential expression in meristematic and vascular tissues. Their YFP-fusion proteins localized primarily to the plasma membrane in root meristem epidermal cells. We selected a mutant line that was considered to be null for each gene. Under normal growth conditions, neither single mutants nor multiple mutants of any combination exhibited noticeable phenotypic changes. However, stress conditions with mannitol or NaCl suppressed main root growth and reduced proximal root meristem size to a greater extent in the pip5k7pip5k8pip5k9 triple mutant than in the wild type. In root meristem epidermal cells of the triple mutant, where plasma membrane localization of the PtdIns(4,5)P2 marker P24Y is impaired to a large extent, brefeldin A body formation is retarded compared with the wild type under hyperosmotic stress. These results indicate that PIP5K7, PIP5K8, and PIP5K9 are not required under normal growth conditions, but are redundantly involved in root growth adaptation to hyperosmotic conditions, possibly through the PtdIns(4,5)P2 function promoting plasma membrane recycling in root meristem cells.

SAP130 and CSN1 interact and regulate male gametogenesis in Arabidopsis thaliana.

COP9 signalosome (CSN) is a nuclear complex composed of eight distinct subunits that governs vast developmental processes in Arabidopsis thaliana (L.) Heynh. The null alleles of csn mutants display pleiotropic phenotypes that result in seedling lethality. To date, several partially complemented transgenic plants, expressing the particular CSN subunit in its corresponding null mutant allele, were utilized to bypass seedling lethality and investigate CSN regulation at later stages of development. One such transgenic plant corresponding to CSN1 subunit, fus6/CSN1-3-4, accumulates wild-type level of CSN1 and displays normal plant architecture at vegetative stage. Here we show through histological analyses that fus6/CSN1-3-4 plants display impairment of pollen development at the bicellular stage. This defect is identical to that observed in RNAi plants of SAP130, encoding a subunit of the multiprotein splicing factor SF3b. We further dissected the previously reported interaction between CSN1 and SAP130, to reveal that approximately 100 amino-acid residues located at the N-terminal end of CSN1 (CSN1NN) were essential for this interaction. In silico structure modeling demonstrated that CSN1NN could swing out towards SAP130 to dock onto its Helical Insertion protruding from the structure. These results support our model that CSN1 embeds itself within CSN protein complex through its C-terminal half and reaches out to targets through its N-terminal portion of the protein. Taken together, this is the first report to document the identical loss-of-function phenotypes of CSN1 and SAP130 during male gametogenesis. Thus, we propose that SAP130 and CSN1 coordinately regulate development of male reproductive organs.

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5-bisphosphate signal on the plasma membrane and attenuates root hair elongation.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] serves as a subcellular signal on the plasma membrane, mediating various cell-polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant-unique plasma membrane protein with phosphoinositide-binding activity, in PtdIns(4,5)P2 signaling for root hair tip growth. The long-root-hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P2 -producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter-driven PCaP2-green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P2 marker 2xCHERRY-2xPHPLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P2 signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide-binding domain PCaP2N23 , root hair-specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2-GFP. Inducibly overexpressed PCaP2-GFP, but not its derivative lacking the PCaP2N23 domain, replaced 2xCHERRY-2xPHPLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4-64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2-GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P2 signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P2 and attenuates root hair elongation.

A Newly Identified Flower-Specific Splice Variant of AUXIN RESPONSE FACTOR8 Regulates Stamen Elongation and Endothecium Lignification in Arabidopsis.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26 Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes.

SNAIL1 is essential for female gametogenesis in Arabidopsis thaliana.

Two yeast Brix family members Ssf1 and Ssf2, involved in large ribosomal subunit synthesis, are essential for yeast cell viability and mating efficiency. Their putative homologs exist in the Arabidopsis genome; however, their role in plant development is unknown. Here, we show that Arabidopsis thaliana SNAIL1 (AtSNAIL1), a protein sharing high sequence identity with yeast Ssf1 and Ssf2, is critical to mitosis progression of female gametophyte development. The snail1 homozygous mutant was nonviable and its heterozygous mutant was semi-sterile with shorter siliques. The mutation in SNAIL1 led to absence of female transmission and reduced male transmission. Further phenotypic analysis showed that the synchronic development of female gametophyte in the snail1 heterozygous mutant was greatly impaired and the snail1 pollen tube growth, in vivo, was also compromised. Furthermore, SNAIL1 was a nucleolar-localized protein with a putative role in protein synthesis. Our data suggest that SNAIL1 may function in ribosome biogenesis like Ssf1 and Ssf2 and plays an important role during megagametogenesis in Arabidopsis.

Aromatic amino acid decarboxylase is involved in volatile phenylacetaldehyde production in loquat (Eriobotrya japonica) flowers.

Post anthesis, loquat flowers emit volatile benzenoids, including phenylacetaldehyde, phenylethyl alcohol, and 2-phenethyl benzoate. Previous studies have shown that pyridoxal phosphate-dependent aromatic L-amino acid decarboxylase (AADC) produces phenylacetaldehyde from L-phenylalanine. Here, two AADC genes (EjAADC1 and EjAADC2) were isolated from loquat (Eriobotrya japonica) flowers. The EjAADC1 and EjAADC2 proteins showed approximately 72% and 55% identity, respectively, to a rose AADC homolog that has phenylacetaldehyde synthase activity. Transcript analyses indicated that EjAADC1 was specifically expressed in petals, with the highest level of expression in fully opened flowers; the petals showed high levels of volatile benzenoids, including phenylacetaldehyde. In contrast, EjAADC2 was expressed at a lower level than EjAADC1 in all tested tissues, including leaves and developing flowers. Functional characterization of a recombinant EjAADC1 protein expressed in Escherichia coli showed that it catalyzes the formation of phenylacetaldehyde from L-phenylalanine in a pyridoxal phosphate-dependent manner. Our results suggest that EjAADC1 is mainly responsible for the biosynthesis of volatile benzenoids in loquat flowers.

Characterization of an O-methyltransferase specific to guaiacol-type benzenoids from the flowers of loquat (Eriobotrya japonica).

Volatile benzenoids, including methyl p-methoxybenzoate, p-anisaldehyde, and p-anisalcohol, are responsible for the sweet and characteristic fragrance of loquat (Eriobotrya japonica, Rosaceae) flowers. Although the full pathway of volatile benzenoid synthesis has yet to be elucidated, their chemical structures suggest that O-methyltransferases are present in loquat and function in the methylation of the para-OH groups. In the present study, we used RNA-sequencing to identify four loquat genes (EjOMT1, EjOMT2, EjOMT3, and EjOMT4) that encode O-methyltransferases. We found that EjOMT1 was highly expressed in floral tissues, with an expression pattern that coincided with changes in intracellular volatile benzenoids during flower development. Recombinant EjOMT1 protein expressed in Escherichia coli showed the highest activity towards guaiacol with a Km value of 35 µM. Furthermore, the protein also showed lesser activities towards guaiacol-type benzenoids including eugenol, isoeugenol, vanillin, and ferulic acid, in addition to much weaker activities towards catechol and p-hydroxybenzenoid derivatives. However, no activity was shown towards phenylpropenes without m-methoxy substitution, t-anol and chavicol. Taken together, our findings indicate that EjOMT1 has a broad substrate specificity towards compounds with both para-OH and meta-OCH3 groups, unlike previously characterized O-methyltransferases for volatile benzenoid/phenylpropanoid biosynthesis.

Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning and functional characterization of p-methoxybenzoic acid carboxyl methyltransferase.

MAIN CONCLUSION: p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid. Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.

RNA Binding Proteins RZ-1B and RZ-1C Play Critical Roles in Regulating Pre-mRNA Splicing and Gene Expression during Development in Arabidopsis.

Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here, we report the functions of two proteins containing RNA recognition motifs, RZ-1B and RZ-1C, in Arabidopsis thaliana. RZ-1B and RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C termini. RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA binding activity of RZ-1C with SR proteins through overexpression of the C terminus of RZ-1C conferred defective phenotypes similar to those observed in rz-1b rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of RZ-1B and RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that RZ-1C directly targeted FLOWERING LOCUS C (FLC), promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

GLABRA2 Directly Suppresses Basic Helix-Loop-Helix Transcription Factor Genes with Diverse Functions in Root Hair Development.

The Arabidopsis thaliana GLABRA2 (GL2) gene encodes a transcription factor involved in the cell differentiation of various epidermal tissues. During root hair pattern formation, GL2 suppresses root hair development in non-hair cells, acting as a node between the gene regulatory networks for cell fate determination and cell differentiation. Despite the importance of GL2 function, its molecular basis remains obscure because the GL2 target genes leading to the network for cell differentiation are unknown. We identified five basic helix-loop-helix (bHLH) transcription factor genes (ROOT HAIR DEFECTIVE6 [RHD6], RHD6-LIKE1 [RSL1], RSL2, Lj-RHL1-LIKE1 [LRL1], and LRL2) as GL2 direct targets using transcriptional and posttranslational induction systems. Chromatin immunoprecipitation analysis confirmed GL2 binding to upstream regions of these genes in planta. Reporter gene analyses showed that these genes are expressed in various stages of root hair development and are suppressed by GL2 in non-hair cells. GL2 promoter-driven GFP fusions of LRL1 and LRL2, but not those of the other bHLH proteins, conferred root hair development on non-hair cells. These results indicate that GL2 directly suppresses bHLH genes with diverse functions in root hair development.

UV-B-Induced CPD Photolyase Gene Expression is Regulated by UVR8-Dependent and -Independent Pathways in Arabidopsis.

Plants have evolved various mechanisms that protect against the harmful effects of UV-B radiation (280-315 nm) on growth and development. Cyclobutane pyrimidine dimer (CPD) photolyase, the repair enzyme for UV-B-induced CPDs, is essential for protecting cells from UV-B radiation. Expression of the CPD photolyase gene (PHR) is controlled by light with various wavelengths including UV-B, but the mechanisms of this regulation remain poorly understood. In this study, we investigated the regulation of PHR expression by light with various wavelengths, in particular low-fluence UV-B radiation (280 nm, 0.2 µmol m(-2) s(-1)), in Arabidopsis thaliana seedlings grown under light-dark cycles for 7 d and then adapted to the dark for 3 d. Low-fluence UV-B radiation induced CPDs but not reactive oxygen species. AtPHR expression was effectively induced by UV-B, UV-A (375 nm) and blue light. Expression induced by UV-A and blue light was predominantly regulated by the cryptochrome-dependent pathway, whereas phytochromes A and B played a minor but noticeable role. Expression induced by UV-B was predominantly regulated by the UVR8-dependent pathway. AtPHR expression was also mediated by a UVR8-independent pathway, which is correlated with CPD accumulation induced by UV-B radiation. These results indicate that Arabidopsis has evolved diverse mechanisms to regulate CPD photolyase expression by multiple photoreceptor signaling pathways, including UVR8-dependent and -independent pathways, as protection against harmful effects of UV-B radiation.

Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

Plants drastically alter their root system architecture to adapt to different underground growth conditions. During phosphate (Pi) deficiency, most plants including Arabidopsis thaliana enhance the development of lateral roots and root hairs, resulting in bushy and hairy roots. To elucidate the signal pathway specific for the root hair elongation response to Pi deficiency, we investigated the expression of type-B phosphatidylinositol phosphate 5-kinase (PIP5K) genes, as a quantitative factor for root hair elongation in Arabidopsis. At young seedling stages, the PIP5K3 and PIP5K4 genes responded to Pi deficiency in steady-state transcript levels via PHR1-binding sequences (P1BSs) in their upstream regions. Both pip5k3 and pip5k4 single mutants, which exhibit short-root-hair phenotypes, remained responsive to Pi deficiency for root hair elongation; however the pip5k3pip5k4 double mutant exhibited shorter root hairs than the single mutants, and lost responsiveness to Pi deficiency at young seedling stages. In the tactical complementation line in which modified PIP5K3 and PIP5K4 genes with base substitutions in their P1BSs were co-introduced into the double mutant, root hairs of young seedlings had normal lengths under Pi-sufficient conditions, but were not responsive to Pi deficiency. From these results, we conclude that a Pi-deficiency signal is transferred to the pathway for root hair elongation via the PIP5K genes.

ADP1 affects plant architecture by regulating local auxin biosynthesis.

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.

TRANSLUCENT GREEN, an ERF family transcription factor, controls water balance in Arabidopsis by activating the expression of aquaporin genes.

Water is the most abundant molecule in almost all living organisms. Aquaporins are channel proteins that play critical roles in controlling the water content of cells. Here, we report the identification of an AP2/EREBP family transcription factor in Arabidopsis thaliana, TRANSLUCENT GREEN (TG), whose overexpression in transgenic plants gave enhanced drought tolerance and vitrified leaves. TG protein is localized in the nucleus, binds DRE and GCC elements in vitro, and acts as a transcriptional activator in yeast cells. Microarray analysis revealed a total of 330 genes regulated by TG, among which five genes encode aquaporins. A transient expression assay showed that TG directly binds to the promoters of three aquaporin genes, such as AtTIP1;1, AtTIP2;3, and AtPIP2;2, indicating that TG directly regulates the expression of these genes. Moreover, overexpression of AtTIP1;1 resulted in vitrified phenotypes in transgenic Arabidopsis plants, similar to those observed in TG overexpression lines. Water injection into wild-type leaves recapitulated the vitrified leaf phenotypes, which was reversed by cutting off the water supply from vascular bundles. Taken together, our data support that TG controls water balance in Arabidopsis through directly activating the expression of aquaporin genes.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.