Subramanian Sankaranarayanan

ABI3 controls embryo degreening through Mendel's I locus

Significance Occurrence of mature green seeds in oil-seed crops such as canola and soybean causes severe losses in revenue. Retention of chlorophyll in seeds can be an undesirable trait as it affects seed maturation, seed oil, and meal quality. We show that the abscisic acid (ABA, plant hormone) dependent transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3), confers seed degreening by regulating Mendel’s stay-green genes. This study unveils a new role for ABI3 in removal of seed chlorophyll in addition to its functions in embryo maturation and conferring desiccation tolerance. This pathway could be manipulated to tackle the cold-induced green seed problem in oil-seed crops. Chlorophyll (chl) is essential for light capture and is the starting point that provides the energy for photosynthesis and thus plant growth. Obviously, for this reason, retention of the green chlorophyll pigment is considered a desirable crop trait. However, the presence of chlorophyll in mature seeds can be an undesirable trait that can affect seed maturation, seed oil quality, and meal quality. Occurrence of mature green seeds in oil crops such as canola and soybean due to unfavorable weather conditions during seed maturity is known to cause severe losses in revenue. One recently identified candidate that controls the chlorophyll degradation machinery is the stay-green gene, SGR1 that was mapped to Mendel’s I locus responsible for cotyledon color (yellow versus green) in peas. A defect in SGR1 leads to leaf stay-green phenotypes in Arabidopsis and rice, but the role of SGR1 in seed degreening and the signaling machinery that converges on SGR1 have remained elusive. To decipher the gene regulatory network that controls degreening in Arabidopsis, we have used an embryo stay-green mutant to demonstrate that embryo degreening is achieved by the SGR family and that this whole process is regulated by the phytohormone abscisic acid (ABA) through ABSCISIC ACID INSENSITIVE 3 (ABI3); a B3 domain transcription factor that has a highly conserved and essential role in seed maturation, conferring desiccation tolerance. Misexpression of ABI3 was sufficient to rescue cold-induced green seed phenotype in Arabidopsis. This finding reveals a mechanistic role for ABI3 during seed degreening and thus targeting of this pathway could provide a solution to the green seed problem in various oil-seed crops.

The AMOR Arabinogalactan Sugar Chain Induces Pollen-Tube Competency to Respond to Ovular Guidance

Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the β isomer of methyl-glucuronosyl galactose (4-Me-GlcA-β-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen

We have previously proposed that Exo70A1 is required in the Brassicaceae stigma to control the early stages of pollen hydration and pollen tube penetration through the stigmatic surface, following compatible pollination. However, recent work has raised questions regarding Arabidopsis thalianaExo70A1’s expression in the stigma and its role in stigma receptivity to compatible pollen. Here, we verified the expression of Exo70A1 in stigmas from three Brassicaceae species and carefully re-examined Exo70A1’s function in the stigmatic papillae. With previous studies showing that high relative humidity can rescue some pollination defects, essentially bypassing the control of pollen hydration by the Brassicaceae dry stigma, the effect of high humidity was investigated on pollinations with the Arabidopsis exo70A1-1 mutant. Pollinations under low relative humidity resulted in a complete failure of wild-type compatible pollen acceptance by the exo70A1-1 mutant stigma as we had previously seen. However, high relative humidity resulted in a partial rescue of the exo70A1-1 stigmatic papillar defect resulting is some wild-type compatible pollen acceptance and seed set. Thus, these results reaffirmed Exo70A1’s proposed role in the stigma regulating compatible pollen hydration and pollen tube entry and demonstrate that high relative humidity can partially bypass these functions.

Glyoxalase Goes Green: The Expanding Roles of Glyoxalase in Plants

The ubiquitous glyoxalase enzymatic pathway is involved in the detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis. The glyoxalase system has been more extensively studied in animals versus plants. Plant glyoxalases have been primarily associated with stress responses and their overexpression is known to impart tolerance to various abiotic stresses. In plants, glyoxalases exist as multigene families, and new roles for glyoxalases in various developmental and signaling pathways have started to emerge. Glyoxalase-based MG detoxification has now been shown to be important for pollination responses. During self-incompatibility response in Brassicaceae, MG is required to target compatibility factors for proteasomal degradation, while accumulation of glyoxalase leads to MG detoxification and efficient pollination. In this review, we discuss the importance of glyoxalase systems and their emerging biological roles in plants.

Deciphering the Stigmatic Transcriptional Landscape of Compatible and Self-Incompatible Pollinations in Brassica napus Reveals a Rapid Stigma Senescence Response Following Compatible Pollination

Dear Editor, Self-incompatibility (SI) is a genetic mechanism through which flowering plants prevent self-pollination to ensure outcrossing and genetic diversity. In Brassica sp., this mechanism is controlled by the self-incompatibility (S) locus, in which, the stigmatic ‘S-locus receptor kinase (SRK)’ recognizes the ‘S-locus cysteine rich protein (SCR)’ from the self-pollen to elicit an active rejection response. This results in blocking of compatibility factors from being delivered to the site of pollen attachment leading to self-pollen rejection (Chapman and Goring, 2010). In contrast, following recognition of compatible signals from the cross-pollen or compatible pollen (CP), the stigma releases its resources such as water and nutrients to the dry pollen so that the pollen tube can germinate and penetrate the stigmatic cuticle leading to successful fertilization. Thus, an incompatible or self-pollen is fully capable of eliciting a compatible response, but is actively rejected before compatible responses can occur. Following landing of self-pollen or cross-pollen on stigmas of Brassica napus (canola), there is a latent period of 30 min when signals are exchanged between the highly lipophilic pollen coat proteins and the stigmatic components. CP tubes can be observed to emerge between 30 and 90 min after initiation of this interaction. Given that stigmas control the outcome of pollen acceptance or rejection, deciphering the transcriptional changes during this latent period would reveal genes involved in compatible and self-incompatible responses. As expected, when self-incompatible W1 canola stigmas were stained with aniline blue to observe pollen tubes, SI-pollinated stigmas lacked any pollen attachment or pollen tubes at 30 min and 6 h after pollination (Figure 1A, right panel). The weakly attached pollen without any positive interactions is washed away during the staining process. Following compatible pollination, although pollen attachment and pollen tubes could be observed at 6 h, at 30 min after pollination, no pollen attachment could be observed (Figure 1A, right panel). This is due to lack of complete adhesion and pollen tube germination at 30 min after pollination. These observations suggest that analyzing the transcriptome changes 0–30 min following SI and compatible pollinations would likely reveal genes that are triggered by pollen landing on the stigma and could represent genes that are required for promoting SI and compatible responses, respectively. To identify the genes that are differentially regulated by SI and compatible pollinations (Figure 1A, left panel), RNA extracted from self-incompatible W1 stigmas, pollinated with self-pollen or cross-pollen for either 15 or 30 min, were compared against RNA from unpollinated (UP) stigmas through transcriptome profiling, using the Agilent 4 × 44K Brassica Gene Expression Microarrays (G2519F). Following normalization, filtering based on P-values (<0.001) and then by two-fold up-regulation in at least one of the microarray experiments, we identified 621 genes that were differentially regulated. Clustering of these genes (Supplemental Table 1) clearly indicated strong up-regulation of multiple genes across all four treatments. This suggested to us that these were likely expressed pollen genes when SI and compatible pollinated stigmas were compared with UP stigmas that lacked any pollen. Utilizing the high sequence similarity between Arabidopsis and Brassica, we identified the orthologous Arabidopsis genes for the 621 canola genes to facilitate further bioinformatic analyses. Following filtering of the pollen genes from the differentially expressed genes in the microarray experiments (see Supplementary Data), the 621 genes were subdivided into stigma genes (287), pollen genes (181), and stigma–pollen genes (153) (Supplemental Table 2). Since the focus of this study was to identify stigmatic genes specifically regulated by SI and CP, we focused our attention on the 287 stigmatic genes. Clustering the stigmatic genes (Supplemental Table 3) revealed a clear pattern of changes between SI and compatible pollination. Heat maps were generated for the subset of genes that were specifically induced or repressed in SI and compatible pollination (Figure 1B–1E and Supplemental Table 4).

Structure-Activity Relation of AMOR Sugar Molecule that Activates Pollen-Tubes for Ovular Guidance

Structure-activity relationship studies of AMOR disaccharide in Torenia fournieri reveal the various residues that are critical for AMOR activity. Successful fertilization in flowering plants depends on the precise directional growth control of pollen tube through the female pistil tissue toward the female gametophyte contained in the ovule for delivery of nonmotile sperm cells. Cys-rich peptides LUREs secreted from the synergid cells on either side of the egg cell act as ovular attractants of pollen tubes. Competency control by the pistil is crucial for the response of pollen tubes to these ovular attractants. We recently reported that ovular 4-O-methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. The beta isomer of the terminal disaccharide 4-O-methyl-glucuronosyl galactose was essential and sufficient for the competency induction. However, critical and noncritical structures in the disaccharide have not been dissected deeply. Herein, we report the synthesis of new AMOR analogs and the structure-activity relationships for AMOR activity in the presence of these synthesized analogs. Removal of 4-O-methyl group or –COOH from the glucuronosyl residue of the disaccharide dramatically reduces AMOR activity. The pyranose backbone of the second sugar of disaccharide is essential for the activity but not hydroxy groups. The role of beta isomer of the disaccharide 4-Me-GlcA-β(1,6)-Gal is very specific for competency control, as there was no difference in effect among the sugar analogs tested for pollen germination. This study represents the first structure-activity relationship study, to our knowledge, of a sugar molecule involved in plant reproduction, which opens a way for modification of the molecule without loss of activity.

Capacitation in Plant and Animal Fertilization

Sexual reproduction relies on the successful fusion of the sperm and egg cell. Despite the vast differences between plants and animals, there are similarities at a molecular level between plant and animal reproduction. While the molecular basis of fertilization has been extensively studied in plants, the process of capacitation has received little attention until recently. Recent research has started to uncover the molecular basis of plant capacitation. Furthermore, recent studies suggest that the key molecules in plants and animal fertilization are functionally conserved. Here, we review new insights for our understanding of capacitation of pollen tube and fertilization in plants and also propose that there are commonalities in the process of sexual reproduction between plants and animals.

A proposed role for selective autophagy in regulating auxin-dependent lateral root development under phosphate starvation in Arabidopsis.

Plants respond to limited soil nutrient availability by inducing more lateral roots (LR) to increase the root surface area. At the cellular level, nutrient starvation triggers the process of autophagy through which bulk degradation of cellular materials is achieved to facilitate nutrient mobilization. Whether there is any link between the cellular autophagy and induction of LR had remained unknown. We recently showed that the S-Domain receptor Kinase (ARK2) and U Box/Armadillo Repeat-Containing E3 ligase (PUB9) module is required for lateral root formation under phosphate starvation in Arabidopsis thaliana.1 We also showed that PUB9 localized to autophagic bodies following either activation by ARK2 or under phosphate starvation and ark2–1/pub9–1 plants displayed lateral root defects with inability to accumulate auxin in the root tips under phosphate starvation.1 Supplementing exogenous auxin was sufficient to rescue the LR defects in ark2–1/pub9–1 mutant. Blocking of autophagic responses in wild-type Arabidopsis also resulted in inhibition of both lateral roots and auxin accumulation in the root tips indicating the importance of autophagy in mediating auxin accumulation under phosphate starved conditions.1 Here, we propose a model for ARK2/AtPUB9 module in regulation of lateral root development via selective autophagy.

Guiding Principles for Live Cell Imaging of Plants Using Confocal Microscopy

Live cell imaging using confocal microscope is an important and popular technique used by biologists to observe and understand biological events occuring in a living cell. It allows simultaneous understanding of the dynamics and functions of many cellular processes in living cells. The principles of live cell imaging are different from that of fixed cell imaging as in live cells, pigments and fluorescent biomolecules are present in their functional state unlike in fixed cell imaging where they are removed. This poses various challenges in live cell imaging, such as maintaining cell viability under high photobleaching conditions and the use of optimal fluorescent components to overcome the artifacts. Given the multitude of advantages of live cell imaging over conventional microscopy, the purpose of this chapter is to provide a basic understanding of the approaches used to visualize plant cells using confocal microscopy, discuss some common challenges encountered during live cell imaging and provide suggestions to overcome them.

Directional Growth for Sperm Delivery

Flowering plants use a polarized projection of the pollen grain called a pollen tube, to precisely deliver two sperm cells to the female gametes, which are deeply buried in the female gametophyte of the ovules for fertilization. The pollen tubes navigate a long route from the stigma, through the transmitting tract over a considerable amount of time before targeting the individual ovules. How is this precise cellular targeting achieved by the pollen tube? A lot of progress has been made toward identifying the sources of guidance cues that the pollen tube receives and the molecules that act as such guidance cues during its journey toward the ovule. In this chapter, we discuss these pollen tube guidance cues and also the latest tools in bio-imaging and microfluidics that would enable us to gain a better understanding of this process of directional growth for sperm delivery.