A splicing site mutation in <Emphasis Type="Italic">BrpFLC1</Emphasis> and repressed expression of <Emphasis Type="Italic">BrpFLC</Emphasis> genes are associated with the early flowering of purple flowering stalk

FLOWERING LOCUS C (FLC), which encodes a MADS-box domain protein, is a flowering repressor involved in the key position of Arabidopsis (Arabidopsis thaliana) flowering network. In Brassica species, several FLC homologues are involved in flowering time like Arabidopsis FLC. Here, we report the analysis of splicing variation in BrpFLC1 and the expression of BrpFLC homologues associated with early flowering of Purple Flowering Stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey). It was indicated that a splice site mutation happened in intron 6 with G to A at the 5′ splice site. Three alternative splicing patterns of BrpFLC1, including the entire exon 6 excluded and 24 bp or 87 bp of intron 6 retained, were identified in Purple Flowering Stalk. But there was only one normal splicing pattern in Pakchoi (Brassica campestris ssp. chinensis var. communis). Northern blotting and semi-quantitative RT-PCR revealed that the expression levels of the three FLC homologues in Purple Flowering Stalk were lower than that in Pakchoi. However, the expression levels of downstream genes, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT), were higher in Purple Flowering Stalk. These results suggest that a natural splicing site mutation in BrpFLC1 gene and repressed expression of all BrpFLC genes contribute significantly to flowering time variation in Purple Flowering Stalk.     

The role of the miR399-<Emphasis Type="Italic">PHO2</Emphasis> module in the regulation of flowering time in response to different ambient temperatures in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.     

Expression and Functional Analysis of the ZCN1(ZmTFL1) Gene, a TERMINAL FLOWER 1 Homologue that Regulates the Vegetative to Reproductive Transition in Maize

TERMINAL FLOWER 1 (TFL1) homologs play critical roles in regulating flowering time and/or maintaining flowering of meristems. In this study, the gene of maize TFL1 ortholog ZmTFL1 (ZCN1) was cloned from both the tropical inbred line CML288 and temperate inbred line Huangzao 4, and the function of ZmTFL1 (ZCN1) was determined during different periods of floral development. Spatial and temporal expression patterns revealed that ZCN1 was predominantly localized in shoot apical meristems that develop into flowers, and only at low levels in leaves. To further identify the role of ZCN1 in floral development of maize, the morphology of shoot apices in maize during floral development was investigated using laser scanning confocal microscopy. Moreover, the relative levels of expression of ZCN1, ZCN8, DLF1, and ZAP1 genes were determined. Over-expression of ZCN1 partially complemented the late flowering phenotype in the tfl1-14 Arabidopsis mutant. Moreover, transgenic Arabidopsis plants exhibited indeterminate inflorescence with increased shoot length and higher numbers of trichomes on leaves. In addition, expression levels of AP1 were significantly down-regulated in 35S::ZCN1 transgenic Arabidopsis plants. These results indicated that ZCN1 as well as its homolog TFL1 in Arabidopsis are involved in the regulation of floral transition in maize.     

The Suppression of WRKY44 by GIGANTEA-miR172 Pathway Is Involved in Drought Response of Arabidopsis thaliana

Water availability is an important environmental factor that controls flowering time. Many plants accelerate flowering under drought conditions, a phenomenon called drought escape. Four pathways are involved in controlling flowering time, but which ones participate in drought escape is not yet known. In this study, plants with loss-of-function mutations of GIGANTEA (GI) and CONSTANS (CO) exhibited abnormal drought-escape phenotypes. The peak mRNA levels of GI and FKF1 (Flavin-binding Kelch domain F box protein 1) and the mRNA levels of CO and FT (Flowering locus T) changed under drought stress. The microRNA factor miRNA172E was up-regulated by drought stress, and its up-regulation was dependent on GI, while other miRNA172s were not. Water-loss analyses indicated that gi mutants were more sensitive while miRNA172 over-expressing (miRNA172-OX) plants were less so to drought stress than wild-type plants. Digital gene expression and real-time PCR analyses showed that WRKY44 was down-regulated by GI and miRNA172. The WRKY44 protein could interact with TOE1 (a target of miRNA172) in a yeast two-hybrid system. We proposed that GI–miRNA172–WRKY44 may regulate drought escape and drought tolerance by affecting sugar signaling in Arabidopsis.     

Overexpression of the Gm<Emphasis Type="Italic">GAL2</Emphasis> Gene Accelerates Flowering in <Emphasis Type="BoldItalic">Arabidopsis</Emphasis>

A soybean MADS box gene GmGAL2 (Glycine max AGAMOUS Like 2), a homolog of AGL11/STK, was investigated in transgenic Arabidopsis lines. Ectopic expression of GmGAL2 in Arabidopsis enhanced flowering, under both long-day and short-day conditions, by promoting expression of key flowering genes, CONSTANS (CO) and FLOWERING LOCUS T (FT), and lowering expression of floral inhibiter FLOWERING LOCUS C (FLC). Moreover, frequency of silique pod set was also lower in transgenic compared to control Arabidopsis plants. RT-PCR results revealed that GmGAL2 was primarily expressed in the flowers and pods of soybean plants, GmGAL2 expressed higher in SD than LD in soybean.     

The dynamics of FLOWERING LOCUS T expression encodes long‐day information

Long days repeatedly enhance the expression of the FLOWERING LOCUS T (FT) gene during the evening and early night. This signal induces flowering despite low FT expression the rest of the day. To investigate whether this temporal behaviour transmits information, plants of Arabidopsis thaliana were exposed to different day–night cycles, including combinations that induced FT expression out of normal hours. Flowering time best correlated with the integral of FT expression over several days, corrected for a higher evening and early night sensitivity to FT. We generated a system to induce FT expression in a leaf removed 8–12 h later. The expression of flowering genes in the apex and flowering required cycles of induction repeated over several days. Evening and early night FT induction was the most effective. The temporal pattern of FT expression encodes information that discriminates long days from other inputs.     

Expression and functional analysis of <Emphasis Type="Italic">NUCLEAR FACTOR</Emphasis>-<Emphasis Type="Italic">Y</Emphasis>, subunit B genes in barley

NUCLEAR FACTOR-Y, subunit B (NF-YB) comprises a multigene family in plants and has been shown to play important roles in growth, development, and response to environmental stress. In this study, five NF-YBs containing the full-length coding region were obtained from barley (Hordeum vulgare) through database sequence analysis, cloning, and sequencing. Sequence alignment and phylogenetic analysis showed that HvNF-YB3 and HvNF-YB1 were clustered with NF-YB2 and NF-YB3 in Arabidopsis, suggesting these NF-YBs are evolutionary and functionally related. To test this hypothesis, HvNF-YB3 and HvNF-YB1 were overexpressed in Arabidopsis. Overexpression of HvNF-YB1 greatly promoted early flowering in Arabidopsis, supporting that HvNF-YB1may have conserved gene function in flowering time control as NF-YB2 and NF-YB3 in Arabidopsis. Overexpression of HvNF-YB3 in Arabidopsis had no effect on flowering time. An analysis of barley single-nucleotide polymorphism (SNP) data, however, revealed a significant association between an HvNF-YB3 SNP and heading date. While it is unknown whether HvNF-YB3 directly contributes to heading date regulation, the results implied that HvNF-YB3 may also have conserved function in flowering time (heading date in barley) control. Further studies are needed to directly verify these gene functions in barley. Barley NF-YBs showed different expression patterns associated with tissue types, developmental stages, and response to different stress treatments, suggesting that barley NF-YBs may be involved in other physiological processes.     

Dynamic localization of the actin-bundling protein cortexillin I during cell migration

The circadian clock in plants regulates many important physiological and biological processes, including leaf movement. We have used an imaging system to genetically screen Arabidopsis seedlings for altered leaf movement with the aim of identifying a circadian clock gene. A total of 285 genes were selected from publicly available microarrays that showed an expression pattern similar to those of the Arabidopsis core oscillator genes. We subsequently isolated 42 homozygous recessive mutants and analyzed their leaf movements. We also analyzed leaf movements of activation tagging mutants that showed altered flowering time. We found that agl6-1D plants, in which AGAMOUS-LIKE 6 (AGL6) was activated by the 35S enhancer, showed a shortened period of leaf movement as well as a high level of ZEITLUPE (ZTL) expression, reduced amplitude of LATE ELONGATED HYPOCOTYL (LHY) expression, and arrhythmic TIMING OF CAB EXPRESSION1 (TOC1)/CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. A shortened period of leaf movement was also seen in 35S-AGL6-myc plants, although 35S-amiRAGL6 plants, transgenic plants overexpressing an artificial miRNA (amiR) targeting AGL6, showed unaltered leaf movement. The amplitude of CHLOROPHYLL A/B BINDING PROTEIN 2 (CAB2) expression, a circadian output gene, was also reduced in agl6-1D plants. Taken together, these results suggest that AGL6 plays a potential role in the regulation of the circadian clock by regulating ZTL mRNA level in Arabidopsis.     

Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri

Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear.     

Overexpression of cinnamyl alcohol dehydrogenase gene from sweetpotato enhances oxidative stress tolerance in transgenic Arabidopsis

Cinnamyl alcohol dehydrogenase (CAD) is the enzyme in the last step of lignin biosynthetic pathway and is involved in the generation of lignin monomers. IbCAD1 gene in sweetpotato (Ipomoea batatas) was identified, and its expression was induced by abiotic stresses based on promoter analysis. In this study, transgenic Arabidopsis plants overexpressing IbCAD1 directed by CaMV 35S promoter were developed to determine the physiological function of IbCAD1. IbCAD1-overexpressing transgenic plants exhibited better plant growth and higher biomass compared to wild type (WT), under normal growth conditions. CAD activity was increased in leaves and roots of transgenic plants. Sinapyl alcohol dehydrogenase activity was induced to a high level in roots, which suggests that IbCAD1 may regulate biosynthesis of syringyl-type (S) lignin. Lignin content was increased in stems and roots of transgenic plants; this increase was in S lignin rather than guaiacyl (G) lignin. Overexpression of IbCAD1 in Arabidopsis resulted in enhanced seed germination rates and tolerance to reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Taken together, our results show that IbCAD1 controls lignin content by biosynthesizing S units and plays an important role in plant responses to oxidative stress.

     

Ectopic expression of a poplar APETALA3-like gene in tobacco causes early flowering and fast growth

A MADS-box gene, designated PtAP3, was isolated from a floral bud cDNA library derived from Populus tomentosa. Analysis by multiple alignments of both nucleotide and amino acid sequences, together with phylogenetic analysis, revealed that PtAP3 is an ortholog of Arabidopsis AP3. Analysis of RNA extracts from vegetative and reproductive tissues of P. tomentosa by RT-PCR indicated that PtAP3 is expressed in roots, stems, leaves and vegetative and floral buds. Notably, the expression of PtAP3 fluctuated during floral bud development between September and February with differences between male and female buds. In the former, a gradual down-regulation during this period, interrupted by a slight up-regulation in December, was followed by a sharper up-regulation on February. In developing female floral buds, expression was stable from September to November, sharply up-regulated in December, and then gradually down-regulated until February. The functional role of PtAP3 was investigated in transgenic tobacco plants. Of 25 transformants, nine displayed an earlier flowering phenotype compared with the wild type plants. Furthermore, transgenic tobacco had faster growth and more leaves than untransformed controls. The traits proved to be heritable between the T0 and T1 generations. Our results demonstrate a regulatory role of the PtAP3 gene during plant flowering and growth and suggest that the gene may be an interesting target for genetic modification to induce early flowering in plants.     

Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, two MYST histone acetyltransferases HAM1 and HAM2 work redundantly to acetylate histone H4 lysine 5 (H4K5ace) in vitro. The double mutant ham1/ham2 is lethal, which suggests the critical role of HAM1 and HAM2 in development. Here, we used an artificial microRNA (amiRNA) strategy in Arabidopsis to uncover a novel function of HAM1 and HAM2. The amiRNA-HAM1/2 transgenic plants showed early flowering and reduced fertility. In addition, they responded normally to photoperiod, gibberellic acid treatment, and vernalization. The expression of flowering-repressor FLOWERING LOCUS C (FLC) and its homologues, MADS-box Affecting Flowering genes 3/4 (MAF3/4), were decreased in amiRNA-HAM1/2 lines. HAM1 overexpression caused late flowering and elevated expression of FLC and MAF3/4. Mutation of FLC almost rescued the late flowering with HAM1 overexpression, which suggests that HAM1 regulation of flowering time depended on FLC. Global H4 acetylation was decreased in amiRNA-HAM1/2 lines, but increased in HAM1-OE lines, which further confirmed the acetyltransferase activity of HAM1 in vivo. Chromatin immunoprecipitation revealed that H4 hyperacetylation and H4K5ace at FLC and MAF3/4 were less abundant in amiRNA-HAM1/2 lines than the wild type, but were enriched in HAM1-OE lines. Thus, HAM1 and HAM2 may affect flowering time by epigenetic modification of FLC and MAF3/4 chromatins at H4K5 acetylation.     

SOBIR1 and AGB1 independently contribute to nonhost resistance to Pyricularia oryzae (syn. Magnaporthe oryzae) in Arabidopsis thaliana

ABSTRACT

Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a disease devastating to rice. We have studied the Arabidopsis-P. oryzae pathosystem as a model system for nonhost resistance (NHR) and found that SOBIR1, but not BAK1, is a positive regulator of NHR to P. oryzae in Arabidopsis. AGB1 is also involved in NHR. However, the genetic interactions between SOBIR1, BAK1, and AGB1 are uncharacterized. In this study, we delineated the genetic interactions between SOBIR1, BAK1, and AGB1 in NHR to P. oryzae in Arabidopsis and found SOBIR1 and AGB1 independently control NHR to P. oryzae in Arabidopsis pen2-1 mutant plants. Furthermore, XLG2, but not TMM, has a positive role in penetration resistance to P. oryzae in Arabidopsis pen2-1 mutant plants. Our study characterized genetic interactions in Arabidopsis NHR.

Abbreviations: PRR: pattern recognition receptor, RLK: receptor-like kinase, RLP: receptor-like protein, BAK1: BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1, BIR1: BAK1-INTERACTING RECEPTOR-LIKE KINASE 1, SOBIR1: SUPPRESSOR OF BIR1-1-1, AGB1: ARABIDOPSIS G PROTEIN ß-SUBUNIT 1, XLG2: EXTRA-LARGE G PROTEIN 2