Genome-wide identification and expression analysis of the CaLAX and CaPIN gene families in pepper (Capsicum annuum L.) under various abiotic stresses and hormone treatments.

Auxin plays key roles in regulating plant growth and development as well as in response to environmental stresses. The intercellular transport of auxin is mediated by the following four gene families: ATP-binding cassette family B (ABCB), auxin resistant1/like aux1 (AUX/LAX), PIN-formed (PIN), and PIN-like (PILS). Here, the latest assembled pepper (Capsicum annuum L.) genome was used to characterise and analyse the CaLAX and CaPIN gene families. Genome-wide investigations into these families, including chromosomal distributions, phytogenic relationships, and intron/exon structures, were performed. In total, 4 CaLAX and 10 CaPIN genes were mapped to 10 chromosomes. Most of these genes exhibited varied tissue-specific expression patterns assessed by quantitative real-time PCR. The expression profiles of the CaLAX and CaPIN genes under various abiotic stresses (salt, drought, and cold), exogenous phytohormones (IAA, 6-BA, ABA, SA, and MeJA), and polar auxin transport inhibitor treatments were evaluated. Most CaLAX and CaPIN genes were altered by abiotic stress at the transcriptional level in both shoots and roots, and many CaLAX and CaPIN genes were regulated by exogenous phytohormones. Our study helps to identify candidate auxin transporter genes and to further analyse their biological functions in pepper development and in its adaptation to environmental stresses.

Genome-wide identification and expression analysis of ClLAX, ClPIN and ClABCB genes families in Citrullus lanatus under various abiotic stresses and grafting.

BACKGROUND: Auxin plays an important role in regulating plant growth and development as well as in the response of plants to abiotic stresses. Auxin is transported by three kinds of major protein families, including the AUXIN RESISTANT 1/LIKE AUX1 (AUX/LAX) influx carriers, the PIN-FORMED (PIN) efflux carriers and the ATP binding cassette B/P-glycoprotein/Multidrug-resistance (ABCB/MDR/PGP) efflux/condition carriers. The biological function of several auxin transporter genes has been well characterized in Arabidopsis thaliana. However, their function in response to exogenous auxin and abiotic stresses in watermelon (Citrullus lanatus. L) remained unknown. RESULTS: Here, the latest updated watermelon genome was used to characterise the ClLAX, ClPIN and ClABCB family genes from watermelon. The genome-wide analysis of the ClLAX, ClPIN and ClABCB family genes, including chromosome localisation, gene structure, and phylogenic relationships, was carried out. Seven ClLAXs, 11 ClPINs and 15 ClABCBs were mapped on 10 watermelon chromosomes. The expression profiles of the ClLAX, ClPIN and ClABCB genes under exogenous indole-3-acetic acid and various abiotic stresses (salt, drought, and cold stresses) treatments were performed by quantitative real-time PCR (qRT-PCR). The transcriptional level of majority ClLAX, ClPIN and ClABCB genes were changed by abiotic stresses in both shoots and roots. We also analysed the expression levels of ClLAX, ClPIN and ClABCB genes in graft response. CONCLUSION: Analysis of the expression patterns of ClLAX, ClPIN and ClABCB genes under salt, drought, cold treatment and grafting response helps us to understand the possible roles of auxin transporter genes in watermelon adaptation to environmental stresses.

Identification and Expression Profiling of the Auxin Response Factors in Capsicum annuum L. under Abiotic Stress and Hormone Treatments.

Auxin response factors (ARFs) play important roles in regulating plant growth and development and response to environmental stress. An exhaustive analysis of the CaARF family was performed using the latest publicly available genome for pepper (Capsicum annuum L.). In total, 22 non-redundant CaARF gene family members in six classes were analyzed, including chromosome locations, gene structures, conserved motifs of proteins, phylogenetic relationships and Subcellular localization. Phylogenetic analysis of the ARFs from pepper (Capsicum annuum L.), tomato (Solanum lycopersicum L.), Arabidopsis and rice (Oryza sativa L.) revealed both similarity and divergence between the four ARF families, and aided in predicting biological functions of the CaARFs. Furthermore, expression profiling of CaARFs was obtained in various organs and tissues using quantitative real-time RT-PCR (qRT-PCR). Expression analysis of these genes was also conducted with various hormones and abiotic treatments using qRT-PCR. Most CaARF genes were regulated by exogenous hormone treatments at the transcriptional level, and many CaARF genes were altered by abiotic stress. Systematic analysis of CaARF genes is imperative to elucidate the roles of CaARF family members in mediating auxin signaling in the adaptation of pepper to a challenging environment.

SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato.

BACKGROUND AND AIMS: Iron is an essential micronutrient for all organisms and its uptake, translocation, distribution and utilization are regulated in a complex manner in plants. FER, isolated from tomato (Solanum lycopersicum), was the first transcription factor involved in the iron homeostasis of higher plants to be identified. A FER defect in the T3238fer mutant drastically downregulates the expression of iron uptake genes, such as ferric-chelate reductase 1 (LeFRO1) and iron-regulated transporter 1 (LeIRT1); however, the molecular mechanism by which FER regulates genes downstream remains unknown. The aim of this work was therefore to identify the gene that interacts with FER to regulate the iron-deficiency response in tomato. METHODS: The homologue of the Arabidopsis Ib subgroup of the basic helix-loop-helix (bHLH) proteins, SlbHLH068, was identified by using the program BLASTP against the AtbHLH39 amino acid sequence in the tomato genome. The interaction between SlbHLH068 and FER was detected using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. In addition, virus-induced gene silencing (VIGS) was used to generate tomato plants in which SlbHLH068 expression was downregulated. The expression of genes was analysed using northern blot hybridization and multiple RT-PCR analysis. Seedlings of wild-type and mutant plants were grown under conditions of different nutrient deficiency. KEY RESULTS: SlbHLH068 is highly upregulated in roots, leaves and stems in response to iron deficiency. An interaction between SlbHLH068 and FER was demonstrated using yeast two-hybrid and BiFC assays. The heterodimer formed by FER with SlbHLH068 directly bound to the promoter of LeFRO1 and activated the expression of its reporter gene in the yeast assay. The downregulation of SlbHLH068 expression by VIGS resulted in a reduction of LeFRO1 and LeIRT1 expression and iron accumulation in leaves and roots. CONCLUSIONS: The results indicate that SlbHLH068, as a putative transcription factor, is involved in iron homeostasis in tomato via an interaction with FER.

Tomato LeTHIC is an Fe-requiring HMP-P synthase involved in thiamine synthesis and regulated by multiple factors.

Thiamine is a key primary metabolite which is necessary for the viability of all organisms. It is a dietary requirement for mammals because only prokaryotes, fungi and plants are thiamine prototrophs. In contrast to the well documented biosynthetic mechanism in bacteria, much remains to be deciphered in plants. In this work, a tomato thiamine-auxotrophic (thiamineless, tl) mutant was characterized. The tl mutant occurs due to inactivation of LeTHIC transcription as a result of insertion of a large unknown DNA fragment in its 5'-untranslated region. Expression of wild-type LeTHIC in tl plants was able to complement the mutant to wild type. LeTHIC possessed the same function as E.cTHIC [an Escherichia coli 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase involved in synthesis of the pyrimidine moiety of thiamine] because expression of LeTHIC rescued THIC-deficient strains of E. coli under culture conditions without thiamine supplementation, suggesting that plants employ a bacteria-like route of pyrimidine moiety synthesis. LeTHIC is an Fe-S cluster protein localized in chloroplasts, and Fe is required for maintenance of its enzyme activity because Fe deficiency resulted in a significant reduction of thiamine content in tomato leaves. Further, we also showed that the expression of LeTHIC is tightly regulated at the transcriptional and post-transcriptional level by multiple factors, such as light, Fe status and thiamine pyrophosphate (TPP)-riboswitch. The results clearly demonstrated that a feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato. Our results provide a new insight into understanding the molecular mechanism of thiamine biosynthesis in plants.

Ectopic expression of Lc differentially regulated anthocyanin biosynthesis in the floral parts of tobacco (Nicotiana tobacum L.) plants.

BACKGROUND: Anthocyanins are the conspicuous pigments of flowering plants and participate in several aspects of plant development and defense, such as seeds and pollens dispersal. Leaf colour (Lc) is the first basic/helix-loop-helix (bHLH) transcription factor controlling anthocyanin biosynthesis isolated from maize (Zea mays L.). Ectopic expression of maize Lc enhanced anthocyanin biosynthesis in many plants including tobacco (Nicotiana tobacum L.). However, the molecular regulatory mechanism of anthocyanin biosynthesis in the different floral parts of tobacco remains largely unknown. Therefore, the molecular and biochemical characterization of anthocyanin biosynthesis were investigated in the flowers of both wild type and Lc-transgenic tobacco plants. RESULTS: At the reproductive stage, with respect to the different parts of the flowers in wild type SR1, the calyxes and the pistils were green, and the petals and the filaments showed light pink pigmentation; the Lc-transgenic tobacco exhibited light red in calyxes and crimson in petals and in filaments respectively. Correspondingly, the total anthocyanin contents (TAC) in calyxes, petals and filaments of Lc-transgenic plants were much higher than that of the counterparts in SR1. Though the TAC in anthers of Lc-transgenic plants was low, it was still significantly higher than that of SR1. SR1 has almost the same TAC in the pistils as Lc-transgenic plants. Consistent with the intense phenotype and the increased TAC, Lc was weakly expressed in the calyxes and strongly expressed in petals and filaments of Lc-transgenic plants, while Lc was not detected in SR1. The expression level of NtAN2 in petals was similar between SR1 and Lc-transgenic lines. In agreement with the expression profile of Lc, both early (NtCHS) and late anthocyanin-biosynthetic genes (NtDFR, NtF3'H, and NtANS) were coordinately up-regulated in the counterparts of flowers. HPLC analysis demonstrated that the cyanidin (Cya) deposition was mainly responsible for the intense pigmentation of Lc-transgenic tobacco. CONCLUSIONS: Ectopic expression of Lc greatly enhanced both early- and late- anthocyanin-biosynthetic gene expression, and therefore resulted in the Cya-based TAC increase in the calyxes, the filaments and the petals in tobacco plants.

Requirement and functional redundancy of Ib subgroup bHLH proteins for iron deficiency responses and uptake in Arabidopsis thaliana.

The Ib subgroup of the bHLH gene family in Arabidopsis contains four members (AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101). AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deficiency induced transcription factor (FIT), directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLH101 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.

The upregulation of NtAN2 expression at low temperature is required for anthocyanin accumulation in juvenile leaves of Lc-transgenic tobacco (Nicotiana tabacum L.).

Anthocyanins often accumulate in plants subjected to environmental stress, including low temperature. However, the molecular regulatory mechanism of anthocyanin biosynthesis at low temperature is largely unknown. Here, tobacco was transformed with a maize anthocyanin regulatory gene Lc driven by AtSPX3 promoter to investigate the effect of Lc upon the anthocyanin-biosynthesis pathway. We found that the anthocyanin-biosynthesis pathway could not be activated in wild type, while Lc-transgenic tobacco lines exhibited purple pigmentation in juvenile leaves at low temperature. Accordingly, the total anthocyanin contents increased specifically in juvenile leaves in Lc-transgenic lines. Transcriptional analysis showed that NtCHS and NtCHI were induced by low temperature in leaves of wild type and transgenic lines. NtDFR was uniquely expressed in Lc-transgenic lines, but its transcript was not detected in wild type, implying that NtDFR expression in tobacco leaves was dependent on Lc. Furthermore, the expression of NtAN2 (regulatory gene) and NtANS (anthocyanidin synthase gene) was coordinately upregulated in Lc-transgenic lines under low temperature, suggesting that both Lc and NtAN2 might activate the expression of NtANS. Based on our findings and previous reports, we postulated that Lc interacted with NtAN2 induced by low-temperature stress and consequently stimulated anthocyanin biosynthesis in juvenile leaves of Lc-transgenic tobacco lines.