NCP2/RHD4/SAC7, SAC6 and SAC8 phosphoinositide phosphatases are required for PtdIns4P and PtdIns(4,5)P2 homeostasis and Arabidopsis development.

Phosphoinositides play important roles in plant growth and development. Several SAC domain phosphoinositide phosphatases have been reported to be important for plant development. Here, we show functional analysis of SUPPRESSOR OF ACTIN 6 (SAC6) to SAC8 in Arabidopsis, a subfamily of phosphoinositide phosphatases containing SAC-domain and two transmembrane motifs. We isolated an Arabidopsis mutant ncp2 that lacked cotyledons in seedling and embryo in pid, a background defective in auxin signaling and transport. NCP2 encodes RHD4/SAC7 phosphoinositide phosphatase. SAC6, SAC7 and SAC8 exhibit overlapping and specific expression patterns in seedling and embryo. The sac6 sac7 embryos either fail to develop into seeds, or have three or four cotyledons. The embryo development of sac7 sac8 and sac6 sac7 sac8 mutants is significantly delayed or lethal, and the seedlings are arrested at early stages. Auxin maxima are decreased in double and triple sac mutants. The contents of PtdIns4P and PtdIns(4,5)P2 in sac6 sac7 and sac7 sac8 mutants are dramatically increased. Protein trafficking of the plasma membrane (PM)-localized protein PIN1 and PIN2 from trans-Golgi network/early endosome back to PM is delayed in sac7 sac8 mutants. These results indicate that SAC6-SAC8 are essential for maintaining homeostasis of PtdIns4P and PtdIns(4,5)P2, and auxin-mediated development in Arabidopsis.

The Circadian Clock Gene, TaPRR1, Is Associated With Yield-Related Traits in Wheat (Triticum aestivum L.).

Timing of flowering is crucial for the transformation from vegetative to reproductive growth in the important food crop, wheat (Triticum aestivum L.). The circadian clock is a central part of photoperiod regulation, with Pseudo-Response Regulators (PRRs) representing key components of circadian networks. However, little is known about the effects of PRR family members on yield-related traits in crop plants. In this study, we identified polymorphisms and haplotypes of TaPRR1, demonstrating that natural variations in TaPRR1 are associated with significant differences in yield-related traits including heading date, plant height and thousand grain weight. TaPRR1-6A-Hapla showed an earlier heading date, advanced by 0.9 to 1.7%. TaPRR1-6B-Hapla and TaPRR1-6D-Hapla displayed reduced plant height and increased thousand grain weight of up to 13.3 to 26.4% and 6.3 to 17.3%, respectively. Subcellular localization and transcriptional activity analysis showed that TaPRR1 is a nuclear localization protein with transcriptional activity controlled by an IR domain. The expression profiles of TaPRR1 genes over a 48-h period were characterized by circadian rhythms, which had two peaks under both short- and long- day conditions. In addition, geographical distribution analysis indicated higher distribution frequencies of TaPRR1-6A-Hapla, TaPRR1-6B-Haplb, and TaPRR1-6D-Haplb in different agro-ecological production zones. Furthermore, analysis of molecular variance of the distribution frequency of TaPRR1 haplotypes suggested significant differences in haplotype distribution frequency between landraces and modern cultivars. Our study provides a basis for in-depth understanding of TaPRR1 function on yield-related traits in wheat, as well as establishing theoretical guidance for wheat molecular marker-assisted breeding.

AtMOB1 Genes Regulate Jasmonate Accumulation and Plant Development.

The MOB1 proteins are highly conserved in yeasts, animals, and plants. Previously, we showed that the Arabidopsis (Arabidopsis thaliana) MOB1A gene (AtMOB1A/NCP1) plays critical roles in auxin-mediated plant development. Here, we report that AtMOB1A and AtMOB1B redundantly and negatively regulate jasmonate (JA) accumulation and function in Arabidopsis development. The two MOB1 genes exhibited similar expression patterns, and the MOB1 proteins displayed similar subcellular localizations and physically interacted in vivo. Furthermore, the atmob1a atmob1b (mob1a/1b) double mutant displayed severe developmental defects, which were much stronger than those of either single mutant. Interestingly, many JA-related genes were up-regulated in mob1a/1b, suggesting that AtMOB1A and AtMOB1B negatively regulate the JA pathways. mob1a/1b plants accumulated more JA and were hypersensitive to exogenous JA treatments. Disruption of MYC2, a key gene in JA signaling, in the mob1a/1b background partially alleviated the root defects and JA hypersensitivity observed in mob1a/1b. Moreover, the expression levels of the MYC2-repressed genes PLT1 and PLT2 were significantly decreased in the mob1a/1b double mutant. Our results showed that MOB1A/1B genetically interact with SIK1 and antagonistically modulate JA-related gene expression. Taken together, our findings indicate that AtMOB1A and AtMOB1B play important roles in regulating JA accumulation and Arabidopsis development.

Arabidopsis AGC protein kinases IREH1 and IRE3 control root skewing.

AGC protein kinases play important roles in plant growth and development. Several AGC kinases in Arabidopsis have been functionally characterized. However, the "AGC Other" subfamily, including IRE, IREH1, IRE3 and IRE4, has not been well understood. Here, we reported that ireh1 mutants displayed a root skewing phenotype, which can be enhanced by ire3 mutation. IREH1 and IRE3 were expressed in roots, consistent with their function in controlling root skewing. The fluorescence intensities of the microtubule marker KNpro:EGFP-MBD were decreased in ireh1, ire3 and ireh1 ire3 mutants compared to wild type. The microtubule arrangements in ireh1 and ireh1 ire3 mutants were also altered. IREH1 physically interacted with IRE3 in vitro and in planta. Thus, our findings demonstrate that IREH1 and IRE3 protein kinases play important roles in controlling root skewing, and maintaining microtubule network in Arabidopsis.

Functional Studies of Heading Date-Related Gene TaPRR73, a Paralog of Ppd1 in Common Wheat.

Photoperiod response-related genes play a crucial role in duration of the plant growth. In this study, we focused on TaPRR73, a paralog of "Green Revolution" gene Ppd1 (TaPRR37). We found that overexpression of the truncated TaPRR73 form lacking part of the N-terminal PR domain in transgenic rice promoted heading under long day conditions. Association analysis in common wheat verified that TaPRR73 was an important agronomic photoperiod response gene that significantly affected heading date and plant height; expression analysis proved that specific alleles of TaPRR73-A1 had highly expressed levels in earlier heading lines; the distribution of haplotypes indicated that one of these alleles had been selected in breeding programs. Our results demonstrated that TaPRR73 contributed to regulation of heading date in wheat and could be useful in wheat breeding and in broadening adaptation of the crop to new regions.

NCP1/AtMOB1A Plays Key Roles in Auxin-Mediated Arabidopsis Development.

MOB1 protein is a core component of the Hippo signaling pathway in animals where it is involved in controlling tissue growth and tumor suppression. Plant MOB1 proteins display high sequence homology to animal MOB1 proteins, but little is known regarding their role in plant growth and development. Herein we report the critical roles of Arabidopsis MOB1 (AtMOB1A) in auxin-mediated development in Arabidopsis. We found that loss-of-function mutations in AtMOB1A completely eliminated the formation of cotyledons when combined with mutations in PINOID (PID), which encodes a Ser/Thr protein kinase that participates in auxin signaling and transport. We showed that atmob1a was fully rescued by its Drosophila counterpart, suggesting functional conservation. The atmob1a pid double mutants phenocopied several well-characterized mutant combinations that are defective in auxin biosynthesis or transport. Moreover, we demonstrated that atmob1a greatly enhanced several other known auxin mutants, suggesting that AtMOB1A plays a key role in auxin-mediated plant development. The atmob1a single mutant displayed defects in early embryogenesis and had shorter root and smaller flowers than wild type plants. AtMOB1A is uniformly expressed in embryos and suspensor cells during embryogenesis, consistent with its role in embryo development. AtMOB1A protein is localized to nucleus, cytoplasm, and associated to plasma membrane, suggesting that it plays roles in these subcellular localizations. Furthermore, we showed that disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling.

DNA methylation pattern of Photoperiod-B1 is associated with photoperiod insensitivity in wheat (Triticum aestivum).

As one of the three key components of the 'Green Revolution', photoperiod insensitivity is vital for improved adaptation of wheat (Triticum aestivum) cultivars to a wider geographical range. Photoperiod-B1a (Ppd-B1a) is one of the major genes that confers photoperiod insensitivity in 'Green Revolution' varieties, and has made a significant contribution to wheat yield improvement. In this study, we investigated the mechanisms underlying the photoperiod insensitivity of Ppd-B1a alleles from an epigenetic perspective using a combination of bisulfite genomic sequencing, orthologous comparative analysis, association analysis, linkage analysis and gene expression analysis. Based on the study of a large collection of wheat germplasm, we report two methylation haplotypes of Ppd-B1 and demonstrate that the higher methylation haplotype (haplotype a) was associated with increased copy numbers and higher expression levels of the Ppd-B1 gene, earlier heading and photoperiod insensitivity. Furthermore, assessment of the distribution frequency of the different methylation haplotypes suggested that the methylation patterns have undergone selection during the wheat breeding process. Our study suggests that DNA methylation in the regulatory region of the Ppd-B1 alleles, which is closely related to copy number variation, plays a significant role in wheat breeding, to confer photoperiod insensitivity and better adaptation to a wider geographical range.

Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat.

Plant cysteine protease (CP) genes are induced by abiotic stresses such as drought, yet their functions remain largely unknown. We isolated the full-length cDNA encoding a Triticum aestivum CP gene, designated TaCP, from wheat by the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that TaCP contains an open reading frame encoding a protein of 362 amino acids, which is 96% identical to barley cysteine protease HvSF42. The TaCP transcript level in wheat seedlings was upregulated during polyethylene glycol (PEG) stress, with a peak appearing around 12 h after treatment. TaCP expression level increased rapidly with NaCl treatment at 48 h. TaCP responded strongly to low temperature (4 degree C) treatment from 1 h post-treatment and reached a peak of about 40-fold at 72 h. However, it showed only a very slight response to abscisic acid (ABA). More than one copy of TaCP was present in each of the three genomes of hexaploid wheat and its diploid donors. TaCP fused with green fluorescent protein (GFP) was located in the plasma membrane of onion epidermis cells. Transgenic Arabidopsis plants overexpressing TaCP showed stronger drought tolerance and higher CP activity under water-stressed conditions than wild-type Arabidopsis plants. The results suggest that TaCP plays a role in tolerance to water deficit.

Discovery, evaluation and distribution of haplotypes of the wheat Ppd-D1 gene.

Ppd-D1 is one of the most potent genes affecting the photoperiod response of wheat (Triticum aestivum). Only two alleles, insensitive Ppd-D1a and sensitive Ppd-D1b, were known previously, and these did not adequately explain the broad adaptation of wheat to photoperiod variation. In this study, five diagnostic molecular markers were employed to identify Ppd-D1 haplotypes in 492 wheat varieties from diverse geographic locations and 55 accessions of Aegilops tauschii, the D genome donor species of wheat. Six Ppd-D1 haplotypes, designated I-VI, were identified. Types II, V and VI were considered to be more ancient and types I, III and IV were considered to be derived from type II. The transcript abundances of the Ppd-D1 haplotypes showed continuous variation, being highest for haplotype I, lowest for haplotype III, and correlating negatively with varietal differences in heading time. These haplotypes also significantly affected other agronomic traits. The distribution frequency of Ppd-D1 haplotypes showed partial correlations with both latitudes and altitudes of wheat cultivation regions. The evolution, expression and distribution of Ppd-D1 haplotypes were consistent evidentially with each other. What was regarded as a pair of alleles in the past can now be considered a series of alleles leading to continuous variation.

[A preliminary study on gene expression profile induced by water stress in wheat (Triticum aestivum L.) seedling].

In the present research, suppression subtractive hybridization (SSH) and high density membrane techniques were employed to analysis genes induced by water stress in wheat seedling at 2-leaf stage. The purpose was to comprehensively understand the genetic bases of drought resistance and to find the key genes related to drought resistance in wheat. A total of 181 positive clones were obtained by screening the SSH library including 1 530 individual recombinant clones. The result of the sequence homologous comparison and function querying showed that 83.2% differentially expressed genes were high homologous with those induced by abiotic or biotic stresses in plant and their functions were to protect the cell directly or indirectly from stress strain. Seventeen differentially expressed ESTs found first were registered in GenBank. After further identifying by reverse Northern, RT-PCR and Northern, the gene expression profile induced by water stress in wheat seedling was preliminarily revealed.