Hong-Qing Ling

Draft genome of the wheat A-genome progenitor Triticum urartu

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGAmAm), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis

Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase (FRO2) and ferrous transporter (IRT1) promoters. Overexpression of FIT with either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.

The tomato fer gene encoding a bHLH protein controls iron-uptake responses in roots.

Iron deficiency is among the most common nutritional disorders in plants. To cope with low iron supply, plants with the exception of the Gramineae increase the solubility and uptake of iron by inducing physiological and developmental alterations including iron reduction, soil acidification, Fe(II) transport and root-hair proliferation (strategy I). The chlorotic tomato fer mutant fails to activate the strategy I. It was shown previously that the fer gene is required in the root. Here, we show that fer plants exhibit root developmental phenotypes after low and sufficient iron nutrition indicating that FER acts irrespective of iron supply. Mutant fer roots displayed lower Leirt1 expression than wild-type roots. We isolated the fer gene by map-based cloning and demonstrate that it encodes a protein containing a basic helix–loop–helix domain. fer is expressed in a cell-specific pattern at the root tip independently from iron supply. Our results suggest that FER may control root physiology and development at a transcriptional level in response to iron supply and thus may be the first identified regulator for iron nutrition in plants.

AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in strategy I plants

ABSTRACTAtbHLH29 of Arabidopsis, encoding a bHLH protein, reveals a high similarity to the tomato FER which is proposed as a transcriptional regulator involved in controlling the iron deficiency responses and the iron uptake in tomato. For identification of its biological functions, AtbHLH29 was introduced into the genome of the tomato FER mutant T3238fer mediated by Agrobacterium tumefaciencs. Transgenic plants were regenerated and the stable integration of AtbHLH29 into their genomes was confirmed by Southern hybridization. Molecular analysis demonstrated that expression of the exogenous AtbHLH29 of Arabidopsis in roots of the FER mutant T3238fer enabled to complement the defect functions of FER. The transgenic plants regained the ability to activate the whole iron deficiency responses and showed normal growth as the wild type under iron-limiting stress. Our transformation data demonstrate that AtbHLH29 is a functional ortholog of the tomato FER and can completely replace FER in controlling the effective iron acquisition in tomato. Except of iron, FER protein was directly or indirectly involved in manganese homeostasis due to that loss functions of FER in T3238fer resulted in strong reduction of Mn content in leaves and the defect function on Mn accumulation in leaves was complemented by expression of AtbHLH29 in the transgenic plants. Identification of the similar biological functions of FER and AtbHLH29, which isolated from two systematically wide-diverged “strategy I” plants, suggests that FER might be a universal gene presented in all strategy I plants in controlling effective iron acquisition system in roots.

OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice

Metallothioneins (MTs) are small, cysteine-rich, metal-binding proteins that may be involved in metal homeostasis and detoxification in both plants and animals. OsMT1a, encoding a type 1 metallothionein, was isolated via suppression subtractive hybridization from Brazilian upland rice (Oryza sativa L. cv. Iapar 9). Expression analysis revealed that OsMT1a predominantly expressed in the roots, and was induced by dehydration. Interestingly, the OsMT1a expression was also induced specifically by Zn2+ treatment. Both transgenic plants and yeasts harboring OsMT1a accumulated more Zn2+ than wild type controls, suggesting OsMT1a is most likely to be involved in zinc homeostasis. Transgenic rice plants overexpressing OsMT1a demonstrated enhanced tolerance to drought. The examination of antioxidant enzyme activities demonstrated that catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) were significantly elevated in transgenic plants. Furthermore, the transcripts of several Zn2+-induced CCCH zinc finger transcription factors accumulated in OsMT1a transgenic plants, suggesting that OsMT1a not only participates directly in ROS scavenging pathway but also regulates expression of the zinc finger transcription factors via the alteration of Zn2+ homeostasis, which leads to improved plant stress tolerance.

Isolation and characterization of Fe(III)-chelate reductase gene LeFRO1 in tomato.

Tomato is a model plant for studying molecular mechanisms of iron uptake and metabolism in strategy I plants (dicots and non-graminaceous monocots). Reduction of ferric to ferrous iron on the root surface is an obligatory process for iron acquisition from soil in these plants. LeFRO1 encoding an Fe(III)-chelate reductase protein was isolated from the tomato genome. We show that expression of LeFRO1 in yeast increases Fe(III)-chelate reductase activity. In a transient expression analysis we found that LeFRO1 protein was targeted on the plasma membrane. LeFRO1 transcript was detected in roots, leaves, cotyledons, flowers and young fruits by RT-PCR analysis. Abundance of LeFRO1 mRNA was much lower in young fruits than in other tissues. The transcription intensity of LeFRO1 in roots is dependent on the iron status whereas it is constitutively expressed in leaves. These results indicate that LeFRO1 is required in roots and shoots as well as in reproductive organs for iron homeostasis and that its transcription in roots and shoots is regulated by different control mechanisms. The expression of LeFRO1 was disrupted in the iron-inefficient mutants chloronerva and T3238fer, indicating that FER and CHLN genes are involved in the regulation of LeFRO1 expression in tomato roots. The differential expression of LeFRO1 and LeIRT1 (an iron-regulated metal transporter gene in tomato) in roots of T3238fer under iron-deficient and -sufficient conditions suggests that the FER gene may regulate expression of LeFRO1 more directly than that of LeIRT1 in tomato roots.

OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice.

Phosphate (Pi) homeostasis in plants is required for plant growth and development, and is achieved by the coordination of Pi acquisition, translocation from roots to shoots, and remobilization within plants. Previous reports have demonstrated that over-expression of OsPHR2 (the homolog of AtPHR1) and knockdown of OsSPX1 result in accumulation of excessive shoot Pi in rice. Here we report that OsPHR2 positively regulates the low-affinity Pi transporter gene OsPT2 by physical interaction and upstream regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2-mediated accumulation of excess shoot Pi. OsSPX1 suppresses the regulation on expression of OsPT2 by OsPHR2 and the accumulation of excess shoot Pi, but it does not suppress induction of OsPT2 or the accumulation of excessive shoot Pi in the Ospho2 mutant. Our data also show that OsSPX1 is a negative regulator of OsPHR2 and is involved in the feedback of Pi-signaling network in roots that is defined by OsPHR2 and OsPHO2. This finding provides new insight into the regulatory mechanism of Pi uptake, translocation, allocation and homeostasis in 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.

Co-Overexpression FIT with AtbHLH38 or AtbHLH39 in Arabidopsis-Enhanced Cadmium Tolerance via Increased Cadmium Sequestration in Roots and Improved Iron Homeostasis of Shoots

Cadmium (Cd) is toxic to plant cells. Under Cd exposure, the plant displayed leaf chlorosis, which is a typical symptom of iron (Fe) deficiency. Interactions of Cd with Fe have been reported. However, the molecular mechanisms of Cd-Fe interactions are not well understood. Here, we showed that FER-like Deficiency Induced Transcripition Factor (FIT), AtbHLH38, and AtbHLH39, three basic helix-loop-helix transcription factors involved in Fe homeostasis in plants, also play important roles in Cd tolerance. The gene expression analysis showed that the expression of FIT, AtbHLH38, and AtbHLH39 was up-regulated in the roots of plants treated with Cd. The plants overexpressing AtbHLH39 and double-overexpressing FIT/AtbHLH38 and FIT/AtbHLH39 exhibited more tolerance to Cd exposure than wild type, whereas no Cd tolerance was observed in plants overexpressing either AtbHLH38 or FIT. Further analysis revealed that co-overexpression of FIT with AtbHLH38 or AtbHLH39 constitutively activated the expression of Heavy Metal Associated3 (HMA3), Metal Tolerance Protein3 (MTP3), Iron Regulated Transporter2 (IRT2), and Iron Regulated Gene2 (IREG2), which are involved in the heavy metal detoxification in Arabidopsis (Arabidopis thaliana). Moreover, co-overexpression of FIT with AtbHLH38 or AtbHLH39 also enhanced the expression of NICOTIANAMINE SYNTHETASE1 (NAS1) and NAS2, resulting in the accumulation of nicotiananamine, a crucial chelator for Fe transportation and homeostasis. Finally, we showed that maintaining high Fe content in shoots under Cd exposure could alleviate the Cd toxicity. Our results provide new insight to understand the molecular mechanisms of Cd tolerance in plants.

Arsenic biotransformation and volatilization in transgenic rice

• Biotransformation of arsenic includes oxidation, reduction, methylation, and conversion to more complex organic arsenicals. Members of the class of arsenite (As(III)) S-adenosylmethyltransferase enzymes catalyze As(III) methylation to a variety of mono-, di-, and trimethylated species, some of which are less toxic than As(III) itself. However, no methyltransferase gene has been identified in plants. • Here, an arsM gene from the soil bacterium Rhodopseudomonas palustris was expressed in Japonica rice (Oryza sativa) cv Nipponbare, and the transgenic rice produced methylated arsenic species, which were measured by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). • Both monomethylarsenate (MAs(V)) and dimethylarsenate (DMAs(V)) were detected in the roots and shoots of transgenic rice. After 12 d exposure to As(III), the transgenic rice gave off 10-fold greater volatile arsenicals. • The present study demonstrates that expression of an arsM gene in rice induces arsenic methylation and volatilization, theoretically providing a potential stratagem for phytoremediation.