Physiological and transcriptional analyses of developmental stages along sugarcane leaf

Abstract

Background: Sugarcane is one of the major crops worldwide. It is cultivated in over 100 countries on 22 million ha. The complex genetic architecture and the lack of a complete genomic sequence in sugarcane hamper the adoption of molecular approaches to study its physiology and to develop new varieties. Investments on the development of new sugarcane varieties have been made to maximize sucrose yield, a trait dependent on photosynthetic capacity. However, detailed studies on sugarcane leaves are scarce. In this work, we report the first molecular and physiological characterization of events taking place along a leaf developmental gradient in sugarcane.

Results: Photosynthetic response to CO2 indicated divergence in photosynthetic capacity based on PEPcase activity, corroborated by activity quantification (both in vivo and in vitro) and distinct levels of carbon discrimination on different segments along leaf length. Additionally, leaf segments had contrasting amount of chlorophyll, nitrogen and sugars. RNA-Seq data indicated a plethora of biochemical pathways differentially expressed along the leaf. Some transcription factors families were enriched on each segment and their putative functions corroborate with the distinct developmental stages. Several genes with higher expression in the middle segment, the one with the highest photosynthetic rates, were identified and their role in sugarcane productivity is discussed. Interestingly, sugarcane leaf segments had a different transcriptional behavior compared to previously published data from maize.

Conclusion: This is the first report of leaf developmental analysis in sugarcane. Our data on sugarcane is another source of information for further studies aiming to understand and/or improve C4 photosynthesis. The segments used in this work were distinct in their physiological status allowing deeper molecular analysis. Although limited in some aspects, the comparison to maize indicates that all data acquired on one C4 species cannot always be easily extrapolated to other species. However, our data indicates that some transcriptional factors were segment-specific and the sugarcane leaf undergoes through the process of suberizarion, photosynthesis establishment and senescence.

Fig. 1

Total carbon quantification (a) and carbon isotope discrimination (b) in sugarcane leaf segments: Base “zero” (B0), Base (B), middle (M) and tip (T) sugarcane leaf segments. Letters indicate statistical significance using ANOVA followed by post hoc Student t-test (n = 5; p ≤ 0.05)

Fig. 2

Changes in activity and protein amount of carboxylation enzymes in sugarcane leaf segments: Base “zero” (B0), Base (B), middle (M) and tip (T). (a) Phosphoenolpyruvate carboxylase (PEPcase). Letters indicate statistical significance using ANOVA followed by post hoc Student t-test (n = 4; p ≤ 0.05); (b) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). White bars represent initial activity and black bars total activity. Lower case letters and capital letters indicate statistical significance using ANOVA followed by post hoc Student t-test (n = 4; p ≤ 0.05) on initial and total activity, respectively. For the immunoblots the same amount of protein (100 μg) was loaded for each sample. Three independent biological replicates are shown for each segment

Fig. 3

Total nitrogen quantification (a) and chlorophyll content (b) in sugarcane leaf segments: Base “zero” (B0), Base (B), middle (M) and tip (T). Letters indicate statistical significance using ANOVA followed by post hoc Student t-test (n = 5; p ≤ 0.05)

Fig. 4

Representation of the RNA-seq contrasts between segments. Each segment was compared against the previous one (basal/distal length) originating three contrast: Base - Base "zero" (B-B0); Middle – Base (M-B); Tip – Middle (T-M). The number of differentially expressed genes (DEG) is depicted under the arrows representing the contrasts. Number of genes overexpressed on each segment considering different contrasts is shown above the graphic bars

Fig. 5

Venn diagram showing the overlap and exclusiveness of genes from each contrast: Base - Base "zero" (B-B0); Middle – Base (M-B); Tip – Middle (T-M)

Fig. 6

Spearman correlation between fifteen segments along developmental gradient of maize leaves (M1 to M15 - published by [48]) and the four sugarcane leaf segments (B0, B, M and T - this study)