Automated stomata detection in oil palm with convolutional neural network.

Stomatal density is an important trait for breeding selection of drought tolerant oil palms; however, its measurement is extremely tedious. To accelerate this process, we developed an automated system. Leaf samples from 128 palms ranging from nursery (1 years old), juvenile (2-3 years old) and mature (> 10 years old) were collected to build an oil palm specific stomata detection model. Micrographs were split into tiles, then used to train a stomata object detection convolutional neural network model through transfer learning. The detection model was then tested on leaf samples acquired from three independent oil palm populations of young seedlings (A), juveniles (B) and productive adults (C). The detection accuracy, measured in precision and recall, was 98.00% and 99.50% for set A, 99.70% and 97.65% for set B, and 99.55% and 99.62% for set C, respectively. The detection model was cross-applied to another set of adult palms using stomata images taken with a different microscope and under different conditions (D), resulting in precision and recall accuracy of 99.72% and 96.88%, respectively. This indicates that the model built generalized well, in addition has high transferability. With the completion of this detection model, stomatal density measurement can be accelerated. This in turn will accelerate the breeding selection for drought tolerance.

Diurnal biomarkers reveal key photosynthetic genes associated with increased oil palm yield.

To investigate limiters of photosynthate assimilation in the carbon-source limited crop, oil palm (Elaeis guineensis Jacq.), we measured differential metabolite, gene expression and the gas exchange in leaves in an open field for palms with distinct mesocarp oil content. We observed higher concentrations of glucose 1-phosphate, glucose 6-phosphate, sucrose 6-phosphate, and sucrose in high-oil content palms with the greatest difference being at 11:00 (p-value ≤0.05) immediately after the period of low morning light intensity. Three important photosynthetic genes were identified using differentially expressed gene analysis (DEGs) and were found to be significantly enriched through Gene Ontology (GO) and pathway enrichment: chlorophyll a-b binding protein (CAB-13), photosystem I (PSI), and Ferredoxin-NADP reductase (FNR), particularly for sampling points at non-peak light (11:00 and 19:00), ranging from 3.3-fold (PSI) and 5.6-fold (FNR) to 10.3-fold (CAB-13). Subsequent gas exchange measurements further supported increased carbon assimilation through higher level of internal CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate (E) in high-oil content palms. The selection for higher expression of key photosynthesis genes together with CO2 assimilation under low light is likely to be important for crop improvement, in particular at full maturity and under high density planting regimes where light competition exists between palms.

Differential gene expression at different stages of mesocarp development in high- and low-yielding oil palm.

BACKGROUND: The oil yield trait of oil palm is expected to involve multiple genes, environmental influences and interactions. Many of the underlying mechanisms that contribute to oil yield are still poorly understood. In this study, we used a microarray approach to study the gene expression profiles of mesocarp tissue at different developmental stages, comparing genetically related high- and low- oil yielding palms to identify genes that contributed to the higher oil-yielding palm and might contribute to the wider genetic improvement of oil palm breeding populations. RESULTS: A total of 3412 (2001 annotated) gene candidates were found to be significantly differentially expressed between high- and low-yielding palms at at least one of the different stages of mesocarp development evaluated. Gene Ontologies (GO) enrichment analysis identified 28 significantly enriched GO terms, including regulation of transcription, fatty acid biosynthesis and metabolic processes. These differentially expressed genes comprise several transcription factors, such as, bHLH, Dof zinc finger proteins and MADS box proteins. Several genes involved in glycolysis, TCA, and fatty acid biosynthesis pathways were also found up-regulated in high-yielding oil palm, among them; pyruvate dehydrogenase E1 component Subunit Beta (PDH), ATP-citrate lyase, ß- ketoacyl-ACP synthases I (KAS I), ß- ketoacyl-ACP synthases III (KAS III) and ketoacyl-ACP reductase (KAR). Sucrose metabolism-related genes such as Invertase, Sucrose Synthase 2 and Sucrose Phosphatase 2 were found to be down-regulated in high-yielding oil palms, compared to the lower yield palms. CONCLUSIONS: Our findings indicate that a higher carbon flux (channeled through down-regulation of the Sucrose Synthase 2 pathway) was being utilized by up-regulated genes involved in glycolysis, TCA and fatty acid biosynthesis leading to enhanced oil production in the high-yielding oil palm. These findings are an important stepping stone to understand the processes that lead to production of high-yielding oil palms and have implications for breeding to maximize oil production.

Hormones, polyamines, and cell wall metabolism during oil palm fruit mesocarp development and ripening.

Oil palm is one of the most productive oil-producing crops and can store up to 90% oil in its fruit mesocarp. Oil palm fruit is a sessile drupe consisting of a fleshy mesocarp from which palm oil is extracted. Biochemical changes in the mesocarp cell walls, polyamines, and hormones at different ripening stages of oil palm fruits were studied, and the relationship between the structural and the biochemical metabolism of oil palm fruits during ripening is discussed. Time-course analysis of the changes in expression of polyamines, hormones, and cell-wall-related genes and metabolites provided insights into the complex processes and interactions involved in fruit development. Overall, a strong reduction in auxin-responsive gene expression was observed from 18 to 22 weeks after pollination. High polyamine concentrations coincided with fruit enlargement during lipid accumulation and latter stages of maturation. The trend of abscisic acid (ABA) concentration was concordant with GA4 but opposite to the GA3 profile such that as ABA levels increase the resulting elevated ABA/GA3 ratio clearly coincides with maturation. Polygalacturonase, expansin, and actin gene expressions were also observed to increase during fruit maturation. The identification of the master regulators of these coordinated processes may allow screening for oil palm variants with altered ripening profiles.

Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array.

Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA) and triacylglycerol (TAG) assembly, along with the tricarboxylic acid cycle (TCA) and glycolysis pathway at 16 Weeks After Anthesis (WAA) exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01), and rice (p-value < 0.01) arrays. The oil palm microarray data also showed comparable correlation of expression (r² = 0.569, p < 0.01) throughout mesocarp development to transcriptome (RNA sequencing) data, and improved correlation over quantitative real-time PCR (qPCR) (r² = 0.721, p < 0.01) of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield.