Muhammad Jamshed

Proteomic Analysis of Brassica Stigmatic Proteins Following the Self-incompatibility Reaction Reveals a Role for Microtubule Dynamics During Pollen Responses

Mate selection and maintenance of genetic diversity is crucial to successful reproduction and species survival. Plants utilize self-incompatibility system as a genetic barrier to prevent self pollen from developing on the pistil, leading to hybrid vigor and diversity. In Brassica (canola, kale, and broccoli), an allele-specific interaction between the pollen SCR/SP11 (S-locus cysteine rich protein/S locus protein 11) and the pistil S Receptor Kinase, results in the activation of SRK which recruits the Arm Repeat Containing 1 (ARC1) E3 ligase to the proteasome. The targets of Arm Repeat Containing 1 are proposed to be compatibility factors, which when targeted for degradation by Arm Repeat Containing 1 results in pollen rejection. Despite the fact that protein degradation is predicted to be important for successful self-pollen rejection, the identity of the various proteins whose abundance is altered by the SI pathway has remained unknown. To identify potential candidate proteins regulated by the SI response, we have used the two-dimensional difference gel electrophoresis analysis, coupled with matrix-assisted laser desorption ionization/time of flight/MS. We identified 56 differential protein spots with 19 unique candidate proteins whose abundance is down-regulated following self-incompatible pollinations. The identified differentials are predicted to function in various pathways including biosynthetic pathways, signaling, cytoskeletal organization, and exocytosis. From the 19 unique proteins identified, we investigated the role of tubulin and the microtubule network during both self-incompatible and compatible pollen responses. Moderate changes in the microtubule network were observed with self-incompatible pollinations; however, a more distinct localized break-down of the microtubule network was observed during compatible pollinations, that is likely mediated by EXO70A1, leading to successful pollination.

Farnesylation mediates brassinosteroid biosynthesis to regulate abscisic acid responses.

Protein farnesylation is a post-translational modification involving the addition of a 15-carbon farnesyl isoprenoid to the carboxy terminus of select proteins1–3. Although the roles of this lipid modification are clear in both fungal and animal signalling, many of the mechanistic functions of farnesylation in plant signalling are still unknown. Here, we show that CYP85A2, the cytochrome P450 enzyme that performs the last step in brassinosteroid biosynthesis (conversion of castasterone to brassinolide)4, must be farnesylated to function in Arabidopsis. Loss of either CYP85A2 or CYP85A2 farnesylation results in reduced brassinolide accumulation and increased plant responsiveness to the hormone abscisic acid (ABA) and overall drought tolerance, explaining previous observations5. This result not only directly links farnesylation to brassinosteroid biosynthesis but also suggests new strategies to maintain crop yield under challenging climatic conditions.

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).

Quantitative Trait Locus Mapping for Verticillium wilt Resistance in an Upland Cotton Recombinant Inbred Line Using SNP-Based High Density Genetic Map

Verticillium wilt (VW) caused by Verticillium dahlia Kleb is one of the most destructive diseases of cotton. Numerous efforts have been made to improve the resistance of upland cotton against VW, with little progress achieved due to the paucity of upland cotton breeding germplasms with high level of resistance to VW. Gossypium barbadense was regarded as more resistant compared to upland cotton; however, it is difficult to apply the resistance from G. barbadense to upland cotton improvement because of the hybrid breakdown and the difficulty to fix resistant phenotype in their interspecific filial. Here we reported QTLs related to VW resistance identified in upland cotton based on 1 year experiment in greenhouse with six replications and 4 years investigations in field with two replications each year. In total, 119 QTLs of disease index (DI) and of disease incidence (DInc) were identified on 25 chromosome of cotton genome except chromosome 13 (c13). For DI, 62 QTLs explaining 3.7–12.2% of the observed phenotypic variations were detected on 24 chromosomes except c11 and c13. For DInc, 59 QTLs explaining 2.3–21.30% of the observed PV were identified on 19 chromosomes except c5, c8, c12-c13, c18-c19, and c26. Seven DI QTLs were detected to be stable in at least environments, among which six have sGK9708 alleles, while 28 DInc QTLs were detected to be stable in at least environments. Eighteen QTL clusters containing 40 QTLs were identified on 13 chromosomes (c1-c4, c6-c7, c10, c14, c17 c20-c22, and c24-c25). Most of the stable QTLs aggregated into these clusters. These QTLs and clusters identification can be an important step toward Verticillium wilt resistant gene cloning in upland cotton and provide useful information to understand the complex genetic bases of Verticillium wilt resistance.

Abiotic Stress signaling in wheat- An inclusive overview of hormonal interactions during abiotic stress responses in wheat

Rapid global warming directly impacts agricultural productivity and poses a major challenge to the present-day agriculture. Recent climate change models predict severe losses in crop production worldwide due to the changing environment, and in wheat, this can be as large as 42 Mt/°C rise in temperature. Although wheat occupies the largest total harvested area (38.8%) among the cereals including rice and maize, its total productivity remains the lowest. The major production losses in wheat are caused more by abiotic stresses such as drought, salinity, and high temperature than by biotic insults. Thus, understanding the effects of these stresses becomes indispensable for wheat improvement programs which have depended mainly on the genetic variations present in the wheat genome through conventional breeding. Notably, recent biotechnological breakthroughs in the understanding of gene functions and access to whole genome sequences have opened new avenues for crop improvement. Despite the availability of such resources in wheat, progress is still limited to the understanding of the stress signaling mechanisms using model plants such as Arabidopsis, rice and Brachypodium and not directly using wheat as the model organism. This review presents an inclusive overview of the phenotypic and physiological changes in wheat due to various abiotic stresses followed by the current state of knowledge on the identified mechanisms of perception and signal transduction in wheat. Specifically, this review provides an in-depth analysis of different hormonal interactions and signaling observed during abiotic stress signaling in wheat.

Farnesylation-mediated subcellular localization is required for CYP85A2 function

ABSTRACT Protein farnesylation refers to the addition of a 15-carbon farnesyl isoprenoid to the cysteine residue of the CaaX motif at the carboxy terminus of target proteins. In spite of its known roles in plant development and abiotic stress tolerance, how these processes are precisely regulated by farnesylation had remained elusive. We recently showed that CYP85A2, the cytochrome P450, which converts castasterone to brassinolide in the last step of brassinosteroid synthesis must be farnesylated in order to function in this pathway. Lack of either CYP85A2 or the farnesylation motif of CYP85A2 resulted in reduced brassinolide accumulation, hypersensitivity to ABA, and increased plant drought tolerance. In this study, we have assessed the influence of the N-terminal secretory signal and the C-terminal CaaX motif of CYP85A2 in mediating CYP85A2 function and targeting to endomembrane compartments. We show that CaaX motif could still target CYPA85A2 in the absence of an intact N-terminal secretory signal to the respective membrane compartments and partially rescue cyp85a2-2 phenotypes. However, in the absence of both the CaaX motif and the secretory signal, CYP85A2 is not targeted to the membranes and becomes unstable.