State Key Laboratory of Plant Cell and Chromosome Engineering

Increasing fertilizer consumption has led to low fertilizer use efficiency and environmental problems. Identifying nutrient-efficient genes will facilitate the breeding of crops with improved fertilizer use efficiency. This research performed a genome-wide sequence analysis of the A (NFYA), B (NFYB), and C (NFYC) subunits of Nuclear Factor Y (NF-Y) in wheat (Triticum aestivum) and further investigated their responses to nitrogen and phosphorus availability in wheat seedlings. Sequence mining together with gene cloning identified 18 NFYAs, 34 NFYBs, and 28 NFYCs. The expression of most NFYAs positively responded to low nitrogen and phosphorus availability. In contrast, microRNA169 negatively responded to low nitrogen and phosphorus availability and degraded NFYAs. Overexpressing TaNFYA-B1, a low-nitrogen- and low-phosphorus-inducible NFYA transcript factor on chromosome 6B, significantly increased both nitrogen and phosphorus uptake and grain yield under differing nitrogen and phosphorus supply levels in a field experiment. The increased nitrogen and phosphorus uptake may have resulted from the fact that that overexpressing TaNFYA-B1 stimulated root development and up-regulated the expression of both nitrate and phosphate transporters in roots. Our results suggest that TaNFYA-B1 plays essential roles in root development and in nitrogen and phosphorus usage in wheat. Furthermore, our results provide new knowledge and valuable gene resources that should be useful in efforts to breed crops targeting high yield with less fertilizer input.

Dominance, semidominance, and recessiveness are important modes of Mendelian inheritance. The phytohormone gibberellin (GA) regulates many plant growth and developmental processes. The previously cloned semidominant GA-insensitive (GAI) genes Reduced height1 (Rht1) and Rht2 in wheat (Triticum aestivum) were the basis of the Green Revolution. However, no completely dominant GAI gene has been cloned. Here, we report the molecular characterization of Rht-B1c, a dominant GAI allele in wheat that confers more extreme characteristics than its incompletely dominant alleles. Rht-B1c is caused by a terminal repeat retrotransposons in miniature insertion in the DELLA domain. Yeast two-hybrid assays showed that Rht-B1c protein fails to interact with GA-INSENSITIVE DWARF1 (GID1), thereby blocking GA responses and resulting in extreme dwarfism and pleiotropic effects. By contrast, Rht-B1b protein only reduces interaction with GID1. Furthermore, we analyzed its functions using near-isogenic lines and examined its molecular mechanisms in transgenic rice. These results indicated that the affinity between GID1 and DELLA proteins is key to regulation of the stability of DELLA proteins, and differential interactions determine dominant and semidominant gene responses to GA.

Rider is a novel and recently active Ty1-copia-like retrotransposon isolated from the T3238fer mutant of tomato. Structurally, it is delimited by a duplication of target sites and contains two long terminal direct repeats and an internal open reading frame, which encodes a Ty1-copia-type polyprotein with characteristic protein domains required for retrotransposition. The family of Rider elements has an intermediate copy number and is scattered in the chromosomes of tomato. Rider family members in the tomato genome share high sequence similarity, but different structural groups were identified (full-size elements, deletion derivatives, and solo LTRs). Southern blot analysis in Solanaceae species showed that Rider was a Lycopersicon-specific element. Sequence analysis revealed that among other plants, two Arabidopsis elements (named as Rider-like 1 and Rider-like 2) are most similar to Rider in both the coding and noncoding regions. RT-PCR analysis indicates that Rider is constitutively expressed in tomato plants. The phylogeny-based parsimony analysis and the sequence substitution analyses of these data suggest that these Rider-like elements originated from a recent introgression of Rider into the tomato genome by horizontal transfer 1-6 million years ago. Considering its transcriptional activity and the recent insertion of the element into at least two genes, Rider is a recently active retrotransposon in the tomato genome.

Two cDNAs, designated MWPL1 and MWPL2, encoding putative pectate lyases (Pel; EC 4.2.2.2), which catalyze the cleavage by β ‐elimination of α ‐1→4‐linked galacturonosyl residues of pectins found mostly in middle lamella and primary cell wall in plants, were isolated from ripening fruit of banana ( Musa acuminata ) and their expressions in fruit during ripening and in response to ethylene were investigated. MWPL1 and MWPL2 encode a single polypeptide of 407 and 454 amino acid residues, respectively. The two cDNAs shared an overall identity of 75% in both nucleotide and deduced amino acid sequences. Sequence comparison of MWPL1 and other plant Pels revealed the homology ranging from 76% with zinnia to 48% with ragweed. Southern analysis indicated that MWPL1 might be present as a single copy gene, and there might be up to two copies of MWPL2 in the banana genome. The two cDNAs were expressed differentially and/or spatially in various banana organs, with female flower and fruit tissues showing accumulation of the MWPL2 transcript, which was not detected in root, pseudostem, leaf, male flower and ovary, whereas the MWPL1 transcript was not detectable in all organs tested. In fruit tissue during ripening, although transcripts of both members were not detectable in unripe preclimacteric fruits, they began to accumulate as ripening progressed and the level remained high thereafter in overripe fruits. However, the magnitude of transcript accumulation differed between the two Pel members, with substantially more abundant MWPL2 than MWPL1 in ripening fruit. Similar differential transcript accumulation was also observed between peel and pulp, where the former was markedly higher than the latter. Expression of both Pel members was also affected by exogenous ethylene, whose presence at 5–100 ppm stimulated accumulation of MWPL1 and MWPL2 transcripts in preclimacteric fruit, suggesting that ethylene may play an important regulatory role in regulating Pel expression during fruit ripening of the banana.

Molecular interaction between powdery mildew fungi and Arabidopsis has been widely used as a model system to study plant immunity. Arabidopsis EDR2 (enhanced disease resistance 2) is a well characterized negative regulator in powdery mildew resistance and mildew-induced cell death. Recently, we showed that a mutation in BSK1 (br-signaling kinase 1), suppressed edr2-mediated disease resistance. (1) And the bsk1-1 single mutant displayed enhanced susceptibility to multiple pathogens, indicating that BSK1 plays important roles in plant immunity. BSK1 is a receptor-like cytoplasmic kinase and localizes on plasma membrane; loss of the membrane localization signaling disrupts BSK1 functions in edr2-mediated resistance. Significantly, BSK1 physically associates with the PAMP receptor FLS2 (flagellin sensing 2) and is required by FLS2-mediated ROS burst. (1) Here we show that disruption of BSK1 membrane localization affects the BSK1-FLS2 interactions, suggesting the membrane association of BSK1 is important for both edr2-mediated signaling and the BSK1-FLS2 complex formation. Previously, it was shown that BSK1 is a substrate of the brassinosteroid (BR) receptor BRI1 (brassinosteroid insensitive 1) and plays critical roles in BR signaling. (2) Further exploration of signaling transductions downstream of BSK1-FLS2 complex will not only shed new light on how BSK1 regulates plant immunity, but may also help to dissect the connections between plant growth and defense.

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

Control of final organ size is a fundamental and core process of development of all multicellular organisms, but the mechanisms that set the final size of determinate organs are largely unknown. In a recent study, we demonstrated that the Mediator complex subunit 25 (MED25/PFT1), which is involved in the transcriptional regulation of gene expression, controls the final size of determinate organs by restricting both cell proliferation and cell expansion. med25 mutants formed large organs with larger and slightly more cells, whereas plants overexpressing MED25 produced small organs due to a reduction in both cell number and cell size. Here, we show that a loss-of-function mutation in the Mediator complex subunit 8 (MED8) causes small flowers as a result of reduced cell expansion. Analysis of the med8 med25-2 double mutant reveals that MED8 acts independently of MED25 to regulate cell expansion and organ growth. Taken together, our findings show that MED8 and MED25 play an important role in regulating organ size. Further identification of upstream and downstream components of MED8 and MED25 will help understand how the Mediator complex is involved in organ size control in plants.

The size of seeds and organs is coordinately determined by cell proliferation and cell expansion, but the mechanisms that set final seed and organ size are largely unknown in plants. In a recent study, we have demonstrated that the plant specific G protein γ subunit (AGG3) promotes seed and organ growth by increasing the period of proliferative growth in Arabidopsis. AGG3 is localized in plasma membrane and interacts with the G protein β subunit (AGB1). Homologs of AGG3 in rice (GS3 and DEP1/qPE9-1) have been identified as important quantitative trait loci for seed size and yield. However, rice GS3 and DEP1 influence seed and organ growth by restricting cell proliferation. Here, we discuss the possible molecular mechanisms by which Arabidopsis AGG3 and its rice homologs GS3 and DEP1 control seed and organ size.

Wheat (Triticum aestivum) genotypes with rye (Secale cereale) 1RS chromosomal translocations are widely used in wheat breeding programs because 1RS carries genes for resistance to several diseases. However, some of the pathogens have evolved into new races that overcome the resistance due to extensive use of cultivars with the resistance genes from rye. Therefore, identification and deployment of new resistance sources with desirable agronomic characteristics are important and urgent. We have used winter rye cultivar German White as a source of genes for desirable traits in wheat improvement. A new genotype named WR04-32 was produced through hybridization and chromosome manipulation between common winter wheat cultivar Xiaoyan 6 and German White. This genotype was highly resistant to a wide spectrum of the wheat stripe rust (Puccinia striiformis f. sp. tritici) and powdery mildew (Blumeria graminis f. sp. tritici) pathotypes prevalent in China. The polymerase chain reaction (PCR) result using EST-STS (expressed sequence tag-site tagged sequence) marker STS WE126 specific to 1RS confirmed 1RS in WR04-32, and it was further proved to be a wheat-rye T2BL·1RS translocation line using sequential genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (FISH) with probes pAs1 and pSc119.2 (or pHvG38). In addition to its resistance to stripe rust and powdery mildew, WR04-32 was genetically stable and had desirable agronomic traits, making it a desirable germplasm for wheat breeding.

Cell fate determination is an important process in multicellular organisms. Plant epidermis is a readily-accessible, well-used model for the study of cell fate determination. Our knowledge of cell fate determination is growing steadily due to genetic and molecular analyses of root hairs, trichomes, and stomata, which are derived from the epidermal cells of roots and aerial tissues. Studies have shown that a large number of factors are involved in the establishment of these cell types, especially members of the basic helix-loop-helix (bHLH) superfamily, which is an important family of transcription factors. In this mini-review, we focus on the role of bHLH transcription factors in cell fate determination in Arabidopsis.

The IAN (immune-associated nucleotide-binding protein) family belongs to a family of AIG1-like GTPases. These functionally uncharacterized GTP-binding proteins have unique structures and are differentially expressed in both vertebrate immune cells and plant cells during antibacterial responses. In mammals, the IANs, as a novel family of T cell receptor-responsive proteins, play critical roles in regulation of thymic development and survival of T lymphocytes through the interaction with Bcl-2 family proteins. The Arabidopsis AIG1 and AIG2, which are first identified IAN proteins, are involved in plant resistance to bacteria. Recent analysis of the expression patterns of Arabidopsis IANs suggests that these IAN proteins may play regulatory roles during plant development and response to both biotic and abiotic stress.

Cultivated six-rowed naked barley (Hordeum vulgare ssp. hexastichon var. nudum Hsü) is the oldest cultivated barley in China. We used 35 simple sequence repeat (SSR) markers selected from seven barley linkage groups to study the genetic diversity, geographical differentiation and evolutionary relationships among 65 H. vulgare ssp. hexastichon landrace accessions collected from the Qinghai-Tibet plateau of China, 25 accessions from Tibet (TB), 20 from Qinghai (QH) and 20 from Ganzi (GZ) prefecture in Sichuan province. At the 35 SSR loci we identified 248 alleles among the 65 accessions, 119 (47.98%) of the alleles being common alleles. We also found that the TB accessions possessed 47 private alleles, about 1.5 times more than the 31 private alleles found in the QH accessions and about 5 times more than 9 private alleles found in the GZ accessions. Generally, the TB accessions showed significantly higher genetic diversity than either the QH or GZ accessions whereas no significant difference in genetic diversity was found between the QH and GZ accessions. Partitioning analysis of genetic diversity showed that about 81% of the total variation was due to within-subgroup diversity and about 19% was clearly accounted for by geographical differentiation among the three subgroups. The distributions of alleles for most loci (71.4%) were significantly different among the three subgroups and geographical differentiation could be found according to the distribution of SSR alleles. Cluster analysis indicated that most of the accessions could be clustered into groups which basically coincided with their geographical distribution. These results suggest that Tibet might be a center of genetic diversity for cultivated barley, the cultivated six-rowed naked barley on the Qinghai-Tibet plateau of China may have evolved in Tibet and spread to Qinghai and then to Ganzi prefecture of Sichuan province.

The orientation of plant root growth is modulated by developmental as well as environmental cues. Among the environmental factors, gravity has been extensively studied because of its overpowering effects in modulating root growth direction. However, our knowledge of the effects of other abiotic signals that influence root growth direction is largely unknown. Recently, we have shown that high salinity can modify root growth direction by inducing rapid amyloplast degradation in root columella cells of Arabidopsis thaliana. By exploiting salt overly sensitive (sos) mutants and PIN2 expression analyses, we have shown that the altered root growth direction in response to salt is mediated by ion disequilibrium and is correlated with PIN2 mRNA abundance and expression and localization of the protein. Our study demonstrates that the SOS pathway may mediate this process. Here we discuss our data from broader perspectives. We propose that salt-induced modification of root growth direction is a salt-avoidance behavior, which is an active adaptive mechanism for plants grown under saline conditions. Furthermore, high salinity also stimulates alteration of gravitropic growth of shoots. These findings illustrate that plants have a fine and sophisticated sensory and communication system that enable plants to dynamically and efficiently cope with rapidly changing environment.

Copper is an essential micronutrient for plant growth and development, and copper transporter plays a pivotal role for keeping copper homeostasis. However, little is known about copper transporters in wheat. Here, we report a novel copper transporter gene family, TaCT1, in common wheat. Three TaCT1 homoeologous genes were isolated and assigned to group 5 chromosomes. Each of the TaCT1 genes (TaCT1-5A, -5B or -5D) possesses 12 transmembrane domains. TaCT1 genes exhibited higher transcript levels in leaf than in root, culm and spikelet. Excess copper down-regulated the transcript levels of TaCT1 and copper deficiency-induced TaCT1 expression. Subcellular experiments localized the TaCT1 to the Golgi apparatus. Yeast expression experiments and virus-induced gene silencing analysis indicated that the TaCT1 functioned in copper transport. Site-directed mutagenesis demonstrated that three amino acid residues, Met(35), Met(38) and Cys(365), are required for TaCT1 function. Phylogenetic and functional analyses suggested that homologous genes shared high similarity with TaCT1 may exist exclusively in monocot plants. Our work reveals a novel wheat gene family encoding major facilitator superfamily (MFS)-type copper transporters, and provides evidence for their functional involvement in promoting copper uptake and keeping copper homeostasis in common wheat.

Abstract Differences between alloplasmic lines and euplasmic controls indicated consistent beneficial effects of Aegilops crassa cytoplasm on common wheats. In general, the agronomic performance of alloplasmic lines was superior to that of euplasmic controls; the significant differences observed were ascribed to nucleus‐cytoplasmic (NC) interactions. A number of useful genetic attributes, for example, high yield, good quality and salt tolerance, were identified. A new NC hybrid variety ‘Xiaoshan 2134’ was bred. Field trials showed that the yield NC heterosis of ‘Xiaoshan 2134’ was 13.9% and the yield of ‘Xiaoshan 2134’ was at least 20% higher than that of a control variety widely grown in North China. The results suggested that Ae. crassa cytoplasm could broaden the genetic base of common wheat and improve common wheat cultivars by utilizing NC heterosis.

Developmental plasticity defines an adaptive mechanism, which plays a fundamental role in plant development and survival. How intrinsic or extrinsic factors are integrated to specify cell fates and subsequent organ and body building of a plant is still poorly understood. By studying developmental plasticity of Arabidopsis root hair in response to salt stress, we have begun to understand more about the basis of cellular plasticity. This paper summarizes our recent paper in which it described salt stress induced plasticity of root epidermis and root hair development in Arabidopsis. Analysis of gene expression of the homeobox transcription factor GLABRA2 (GL2), which determines hair/non-hair cell fate, showed that salt stress modulates root epidermal cell proliferation and changes the cell fate decisions. Furthermore, by analyzing the salt overly sensitive (sos) mutants, we showed that salt-induced root hair plastic response is caused by ion disequilibrium and it appears to be adaptive mechanism. Based on the most recent discoveries, we propose here that chromatin remodeling and epigenetic control may be the basis for cell fate changes and the ultimately adaptive plasticity in response to transient changes of environmental conditions.

Abstract Significant progress has been made in understanding the structure of high molecular weight (HMW) glutenin subunits and their role in determining the end use quality of wheat grains. However, few reports have dealt with the development and characterization of knock out mutants for HMW glutenin subunit genes. Here, the molecular analysis of MB14 , a mutant derived from an elite Chinese wheat variety Xiaoyan 54 through chemical mutagenesis is described. SDS‐PAGE and Western blot experiments revealed that, in the seeds of homozygous MB14 plants, the expression of the 1Bx14 subunit was specifically blocked whereas the remaining four subunits (1Ax1, 1By15, 1Dx2, 1Dy12) accumulated to levels comparable to those in the wild type plants. The 5′‐flanking region and the open reading frame (ORF) of the mutant 1Bx14 allele were amplified and compared to the corresponding regions of wild type 1Bx14 . The nucleotide sequences of the 5′‐flanking regions from the mutant and wild type 1Bx14 alleles were identical. However, the ORF of the mutant allele differed from that of the wild type 1Bx14 by three point substitutions, one of which resulted in a premature stop codon in the mutant ORF. Interestingly, the mutant 1Bx14 allele was still transcribed in the developing seeds, but no truncated translation product could be detected by Western blot analysis. Potential application of the 1Bx14 knock out mutant in studying the biological function of 1Bx14 and its contribution to the end use quality control in hexaploid wheat is discussed.

Root apical meristem (RAM) is central for indeterminate growth of plant roots and in sensing environmental stimuli, such as water status. Recently, we reported that PEG8000-simulated mild and moderate osmotic stress induces premature differentiation of RAM, which is a conserved adaptive mechanism in higher plants to cope with water stress. Microarray data analysis revealed that the ABA signaling pathway may be involved in water stress-induced RAM premature differentiation. Here we showed that in wheat, ABA contents increased under water stress with the highest level of ABA in the RAM. Exogenous ABA also induces RAM premature differentiation in both wheat and Arabidopsis plants. Further genetic analysis revealed that loss of function mutations in ABA2 and ABA receptors significantly reduced the level of root tip swelling and RAM premature differentiation in response to PEG-simulated water stress. Together, the results suggest that ABA participates in regulation of PEG-mediated premature differentiation of RAM.

Cytokinin and auxin antagonistically affect cell proliferation and differentiation and thus regulate root meristem size by influencing the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects auxin distribution in the root meristem by repressing the auxin-inducible expression of PIN-FORMED (PIN) auxin transport genes. The PLETHORA (PLT1/2) genes influence root meristem growth by promoting stem cells and transit-amplifying cells. However, the factors connecting cytokinin, auxin, SHY2 and PLT1/2 are largely unknown. In a recent study, we have shown that the DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect root meristem size. Here, we discuss the possible molecular mechanisms by which Arabidopsis DAR2 controls root meristem size.

Powdery mildew pathogens are biotrophic fungi that infect large number of plant species. EDR1 (ENHANCED DISEASE RESISTANCE 1) is a negative regulator of plant disease resistance, and loss-of-function in the EDR1 gene confers enhanced disease resistance to powdery mildew pathogen Golovinomyces cichoracearum. In an edr1 suppressor screen, we recently found that a mutation in HPR1, a component of the THO/TREX complex, suppresses edr1-mediated disease resistance, however the hpr1 mutation enhances the ethylene-induced senescence in edr1. The hpr1 single mutant displays enhanced susceptibility, indicating that HPR1 is involved in plant defense responses. THO/TREX is a conserved protein complex that functions in pre-mRNA processing and mRNA export. Several components of THO/TREX complex in Arabidopsis have been identified. By searching Arabidopsis database, we found that Arabidopsis (Columbia-0) has two copies of UAP56, another component of the THO/TREX complex, and the UAP56 proteins are highly conserved. Similar to human UAP56 protein, Arabidopsis UAP56 also localizes to the nucleus, showing a pattern similar to the splicing speckles. Further characterization of the components of THO/TREX in Arabidopsis will provide new insights into the role of THO/TREX in defense responses in plants.