Two Faces of One Seed: Hormonal Regulation of Dormancy and Germination.

Seed plants have evolved to maintain the dormancy of freshly matured seeds until the appropriate time for germination. Seed dormancy and germination are distinct physiological processes, and the transition from dormancy to germination is not only a critical developmental step in the life cycle of plants but is also important for agricultural production. These processes are precisely regulated by diverse endogenous hormones and environmental cues. Although ABA (abscisic acid) and GAs (gibberellins) are known to be the primary phytohormones that antagonistically regulate seed dormancy, recent findings demonstrate that another phytohormone, auxin, is also critical for inducing and maintaining seed dormancy, and therefore might act as a key protector of seed dormancy. In this review, we summarize our current understanding of the sophisticated molecular networks involving the critical roles of phytohormones in regulating seed dormancy and germination, in which AP2-domain-containing transcription factors play key roles. We also discuss the interactions (crosstalk) of diverse hormonal signals in seed dormancy and germination, focusing on the ABA/GA balance that constitutes the central node.

Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre-existing and induced defence in rice.

Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss-of-function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild-type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1-overexpressing (GIF1-OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild-type plants. More importantly, higher levels of callose were deposited in GIF1-OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1-OE plants when compared with the wild-type plants. We also found that defence-related genes were constitutively activated in the GIF1-OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence.

Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis.

The transition from dormancy to germination in seeds is a key physiological process during the lifecycle of plants. Abscisic acid (ABA) is the sole plant hormone known to maintain seed dormancy; it acts through a gene expression network involving the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3). However, whether other phytohormone pathways function in the maintenance of seed dormancy in response to environmental and internal signals remains an important question. Here, we show that the plant growth hormone auxin, which acts as a versatile trigger in many developmental processes, also plays a critical role in seed dormancy in Arabidopsis. We show that disruptions in auxin signaling in MIR160-overexpressing plants, auxin receptor mutants, or auxin biosynthesis mutants dramatically release seed dormancy, whereas increases in auxin signaling or biosynthesis greatly enhance seed dormancy. Auxin action in seed dormancy requires the ABA signaling pathway (and vice versa), indicating that the roles of auxin and ABA in seed dormancy are interdependent. Furthermore, we show that auxin acts upstream of the major regulator of seed dormancy, ABI3, by recruiting the auxin response factors AUXIN RESPONSE FACTOR 10 and AUXIN RESPONSE FACTOR 16 to control the expression of ABI3 during seed germination. Our study, thus, uncovers a previously unrecognized regulatory factor of seed dormancy and a coordinating network of auxin and ABA signaling in this important process.

Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis.

Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 °C to 30 °C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 °C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.

Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development.

The balance between cell proliferation and cell differentiation is essential for leaf patterning. However, identification of the factors coordinating leaf patterning and cell growth behavior is challenging. Here, we characterized a temperature-sensitive Arabidopsis mutant with leaf blade and venation defects. We mapped the mutation to the sub-2 allele of the SCRAMBLED/STRUBBELIG (SCM/SUB) receptor-like kinase gene whose functions in leaf development have not been demonstrated. The sub-2 mutant displayed impaired blade development, asymmetric leaf shape and altered venation patterning under high ambient temperature (30°C), but these defects were less pronounced at normal growth temperature (22°C). Loss of SCM/SUB function results in reduced cell proliferation and abnormal cell expansion, as well as altered auxin patterning. SCM/SUB is initially expressed throughout leaf primordia and becomes restricted to the vascular cells, coinciding with its roles in early leaf patterning and venation formation. Furthermore, constitutive expression of the SCM/SUB gene also restricts organ growth by inhibiting the transition from cell proliferation to expansion. We propose the existence of a SCM/SUB-mediated developmental stage-specific signal for leaf patterning, and highlight the importance of the balance between cell proliferation and differentiation for leaf morphogenesis.

Arabidopsis acetyl-amido synthetase GH3.5 involvement in camalexin biosynthesis through conjugation of indole-3-carboxylic acid and cysteine and upregulation of camalexin biosynthesis genes.

Camalexin (3-thiazol-2'-yl-indole) is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole-3-acetonitrile (IAN) is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Arabidopsis GH3.5 protein, a multifunctional acetyl-amido synthetase, is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate (ICA(Cys)) in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D. Dihydrocamalexic acid (DHCA), the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5-1D/pad3-1, suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5-1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys), and upregulation of the major biosynthetic pathway genes.

A novel protein RLS1 with NB-ARM domains is involved in chloroplast degradation during leaf senescence in rice.

Leaf senescence, a type of programmed cell death (PCD) characterized by chlorophyll degradation, is important to plant growth and crop productivity. It emerges that autophagy is involved in chloroplast degradation during leaf senescence. However, the molecular mechanism(s) involved in the process is not well understood. In this study, the genetic and physiological characteristics of the rice rls1 (rapid leaf senescence 1) mutant were identified. The rls1 mutant developed small, yellow-brown lesions resembling disease scattered over the whole surfaces of leaves that displayed earlier senescence than those of wild-type plants. The rapid loss of chlorophyll content during senescence was the main cause of accelerated leaf senescence in rls1. Microscopic observation indicated that PCD was misregulated, probably resulting in the accelerated degradation of chloroplasts in rls1 leaves. Map-based cloning of the RLS1 gene revealed that it encodes a previously uncharacterized NB (nucleotide-binding site)-containing protein with an ARM (armadillo) domain at the carboxyl terminus. Consistent with its involvement in leaf senescence, RLS1 was up-regulated during dark-induced leaf senescence and down-regulated by cytokinin. Intriguingly, constitutive expression of RLS1 also slightly accelerated leaf senescence with decreased chlorophyll content in transgenic rice plants. Our study identified a previously uncharacterized NB-ARM protein involved in PCD during plant growth and development, providing a unique tool for dissecting possible autophagy-mediated PCD during senescence in plants.

Plasma membrane localization and potential endocytosis of constitutively expressed XA21 proteins in transgenic rice.

The rice pattern recognition receptor (PRR) XA21 confers race-specific resistance in leaf infection by bacterial blight Xathomonas oryzae pv. oryzae (Xoo), and was shown to be primarily localized to the endoplasmic reticulum (ER) when expressed with its native promoter or overexpressed in the protoplast. However, whether the protein is still ER-localization in the intact cell when overexpressed remains to be identified. Here, we showed that XA21, its kinase-dead mutant XA21P(K736EP), and the triple autophosphorylation mutant XA21P(S686A/T688A/S699A) GFP fusions were primarily localized to the plasma membrane (PM) when overexpressed in the intact transgenic rice cell, and also localized to the ER in the transgenic protoplast. The transgenic plants constitutively expressing the wild-type XA21 or its GFP fusion displayed race-specific resistance to Xoo at the adult and seedling stages. XA21 and XA21P(K736EP) could be internalized probably via the SCAMP-positive early endosomal compartment in the protoplast, suggesting that XA21 might be endocytosed to initiate resistance responses during pathogen infection. We also established a root infection system and demonstrated that XA21 also mediated race-specific resistance responses to Xoo in the root. Our current study provides an insight into the nature of the XA21-mediated resistance and a practical approach using the root cell system to further dissect the cellular signaling of the PRR during the rice-Xoo interaction.

Altered disease development in the eui mutants and Eui overexpressors indicates that gibberellins negatively regulate rice basal disease resistance.

Gibberellins (GAs) form a group of important plant tetracyclic diterpenoid hormones that are involved in many aspects of plant growth and development. Emerging evidence implicates that GAs also play roles in stress responses. However, the role of GAs in biotic stress is largely unknown. Here, we report that knockout or overexpression of the Elongated uppermost internode (Eui) gene encoding a GA deactivating enzyme compromises or increases, respectively, disease resistance to bacterial blight (Xanthomonas oryzae pv. oyrzae) and rice blast (Magnaporthe oryzae). Exogenous application of GA(3) and the inhibitor of GA synthesis (uniconazol) could increase disease susceptibility and resistance, respectively, to bacterial blight. Similarly, uniconazol restored disease resistance of the eui mutant and GA(3) decreased disease resistance of the Eui overexpressors to bacterial blight. Therefore, the change of resistance attributes to GA levels. In consistency with this, the GA metabolism genes OsGA20ox2 and OsGA2ox1 were down-regulated during pathogen challenge. We also found that PR1a induction was enhanced but the SA level was decreased in the Eui overexpressor, while the JA level was reduced in the eui mutant. Together, our current study indicates that GAs play a negative role in rice basal disease resistance, with EUI as a positive modulator through regulating the level of bioactive GAs.

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection.

RNA interference (RNAi) silences gene expression by guiding mRNA degradation in a sequence-specific fashion. Small interfering RNA (siRNA), an intermediate of the RNAi pathway, has been shown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells. Here, we report that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) could inhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-associated 126 kDa protein in intact plant tissue. Our results indicate that transiently expressed shRNA efficiently interfered with TMV infection. The interference observed is sequence-specific, and time- and site-dependent. Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumber mosaic virus (CMV), an unrelated tobamovirus. In order to interfere with TMV accumulation in tobacco leaves, it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation. Our results support the view that RNAi opens the door for novel therapeutic procedures against virus diseases. We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expression could be employed as a potent antiviral treatment in plants.nt antiviral treatment in plants.

Stress-induced Rim2/Hipa pseudogene of rice exhibits alternative tailing and splicing during transcription.

The rice Rim2/Hipa is a unique stress-induced transposon superfamily recently identified in Oryza genomes. In the present study, we conducted genome-wide screening of full-length Rim2 cDNA from the pathogen-induced cDNA libraries and mining of cDNA databases. Four indica and two japonica types of transcripts were identified, which were transcribed from the same Rim2 pseudogene Rim2-42 that contains premature stop codons in the TNP2-TPase coding region. These data demonstrated that the processing of the Rim2 transcripts exhibited variations within and between the two subspecies. These transcripts were found to be produced by alternative transcription (tailing) or splicing from Rim2-42 under stress conditions. An additional Rim2-like transcript (Rim2-XET), a chimera of Rim2 and XET genes, were also found to be derived from read-through. These results show that the Rim2 transposon probably loses its transposition capacity during evolution, and that Rim2-42 inserts downstream of the stress-inducible XET promoter, resulting in Rim2 transcript accumulation upon pathogen attack.

[Construction of an activation tagging library of Arabidopsis and cloning for mutant genes].

Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. In this work, a library of Arabidopsis mutants was constructed by in planta transformation mediated by Agrobacterium tumefaciens containing an activation tagging vector pSKI015 with herbicide Basta as a selection marker (Fig. 1). Among 20000 independent transformants, 38 lines, i.e. about 0.2% of T(1) progeny, show visible morphological phenotypic variations (Fig. 2). Results of Southern blot analysis revealed that most of the transformants have more than three copies of T-DNA insertion (Fig. 3). Plasmid rescue and TAIL-PCR were used to recover the flanking genomic sequences of mutated target genes as the first step towards mutant gene cloning (Fig. 4, 5).

A viral protein suppresses siRNA-directed interference in tobacco mosaic virus infection.

Plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptive defense response that limits virus replication and its spread in plants. The helper component proteinase (HC-Pro) of the potato virus A (PVA, genus Potyvirus) suppresses PTGS of silenced transgenes. Here, the effect of HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using a transient Agrobacterium tumefaciens-based delivery system in intact tissues. It was shown that the interference effect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic and hypersensitive hosts. In the system host, all plants agro-infiltrated with HC-Pro plus siRNA constructs displayed the same symptoms as the negative control. Meanwhile, TMV RNA accumulation was found to be abundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot assays. On the contrary, plants agro-infiltrated with the siRNA construct alone were free of symptoms. Therefore, our study suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses against TMV infection.

[sdt1, a novel Arabidopsis mutant tolerant to drought and high-salinity stresses].

Among the 20000 independent Arabidopsis activation tagging lines, we screened a novel mutant with high-salinity and drought tolerance, namely sdt1 (high-salinity and drought tolerant 1). sdt1 kept normal growth under drought stress by consecutively withholding water for 26d, while all the wild type wilted to death. Furthermore, the same result was repeated under high-salinity stress when sdtl and wild type were treated with 150 mmol/L NaCl. Seeds germination test on the medium indicated that the response of sdt1 to endogenous or exogenous ABA was reduced as compared with wild type. Under drought stress conditions the rates of water loss of sdt1 rosette leaves were significantly lower than that of wild type. Southern hybridization of sdt1 and the result of high-salinity tolerance genetic analysis showed that two copies of T-DNA were inserted into the two linking sites by shoulder to shoulder.

[Physiological and genetic analysis of lesion resembling disease mutants (lrd) of Oryza sativa L].

Ten indica rice and eight japonica rice mutants with lesion resembling disease (lrd27-44) were obtained by gamma-ray mutagenesis of the whole genomes. These mutants exhibited diverse lesion mimic phenotypes under different growth environments, could be accordingly classified two types, sensitive and insensitive to environments. Basing on difference in development of lesion mimics, they can be divided into three categories: whole life lesion mimics (WLLM), vegetative initiation lesion mimics (VILM), and reproductive initiation lesion mimics (RILM). Lesion mimics resulted from the programmed cell death and were triggered by light, but not by wounding. The genetic analysis showed that four mutants, lrd32, lrd39, lrd40 and lrd42, were controlled by one or two recessive loci. Among the 18 lrd mutants, lrd37 and lrd40 conferred non-race-specific resistance to Xanthomonas oryzae pv. oryzae. Gene mapping and cloning of Lrd32 and Lrd40 are under way.

Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice.

Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPH-oxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell.