Selection and Evaluation of a Thornless and HLB-Tolerant Bud-Sport of Pummelo Citrus With an Emphasis on Molecular Mechanisms

The selection of elite bud-sports is an important breeding approach in horticulture. We discovered and evaluated a thornless pummelo bud-sport (TL) that grew more vigorously and was more tolerant to Huanglongbing (HLB) than the thorny wild type (W). To reveal the underlying molecular mechanisms, we carried out whole-genome sequencing of W, and transcriptome comparisons of W, TL, and partially recovered thorny “mutants” (T). The results showed W, TL, and T varied in gene expression, allelic expression, and alternative splicing. Most genes/pathways with significantly altered expression in TL compared to W remained similarly altered in T. Pathway and gene ontology enrichment analysis revealed that the expression of multiple pathways, including photosynthesis and cell wall biosynthesis, was altered among the three genotypes. Remarkably, two polar auxin transporter genes, PIN7 and LAX3, were expressed at a significantly lower level in TL than in both W and T, implying alternation of polar auxin transport in TL may be responsible for the vigorous growth and thornless phenotype. Furthermore, 131 and 68 plant defense-related genes were significantly upregulated and downregulated, respectively, in TL and T compared with W. These genes may be involved in enhanced salicylic acid (SA) dependent defense and repression of defense inducing callose deposition and programmed cell death. Overall, these results indicated that the phenotype changes of the TL bud-sport were associated with tremendous transcriptome alterations, providing new clues and targets for breeding and gene editing for citrus improvement.

Characterization of auxin transporter AUX, PIN and PILS gene families in pineapple and evaluation of expression profiles during reproductive development and under abiotic stresses

Polar auxin transport in plant is mediated by influx and efflux transporters, which are encoded by AUX/LAX, PIN and PILS genes, respectively. The auxin transporter gene families have been characterized in several species from monocots and eudicots. However, a genome-wide overview of auxin transporter gene families in pineapple is not yet available. In this study, we identified a total of threeAcAUX genes, 12 AcPIN genes, and seven AcPILS genes in the pineapple genome, which were variably located on 15 chromosomes. The exon-intron structure of these genes and properties of deduced proteins were relatively conserved within the same family. Most protein motifs were widespread in the AUX, PIN or PILS proteins, whereas a few motifs were absent in only one or two proteins. Analysis of the expression profiles of these genes elucidated that several genes exhibited either preferential or tissue-specific expression patterns in vegetative and/or reproductive tissues. AcAUX2 was specifically expressed in the early developmental ovules, while AcPIN1b and AcPILS2 were strongly expressed in stamens and ovules. AcPIN9b, AcPILS1, AcPILS6a, 6b and 6c were abundantly expressed in stamens. Furthermore, qRT-PCR results showed that several genes in these families were responsive to various abiotic stresses. Comparative analysis indicated that the genes with close evolutionary relationships among pineapple, rice and Arabidopsis exhibited similar expression patterns. Overexpression of the AcAUX1 in Arabidopsis rescued the phenotype in aux1-T, and resulted in increased lateral roots in WT. These results will provide new insights into auxin transporter genes of pineapple and facilitate our understanding of their roles in pineapple growth and development.

Lyso-Phosphatidic Acid Acyl-Transferases: a link with intracellular protein transport in Arabidopsis root cells?

Phosphatidic acid (PA) and Lysophosphatidic acid acyltransferases (LPAATs) might be critical for the secretory pathway. Four extra-plastidial LPAATs (numbered 2,3,4 and 5) were identified in A. thaliana. These AtLPAATs, displaying an enzymatic activity specific for LPA to produce PA, are located in the endomembrane system. We focused on the putative role of the AtLPAATs 3, 4 and 5 in the secretory pathway of root cells through genetical (knock-out mutants), biochemical (activity inhibitor, lipid analyses) and imaging (live and immuno-confocal microscopies) approaches. Treating a lpaat4;lpaat5 double mutant with the LPAAT inhibitor CI976 showed a primary root growth decrease. The transport of the auxin transporter PIN2 was disturbed in this lpaat4;lpaat5 double mutant treated with CI976, but not that of H+-ATPases. The lpaat4;lpaat5 double mutant was sensitive to salt stress and the transport of the aquaporin PIP2;7 to the plasma membrane in the lpaat4;lpaat5 double mutant treated with CI976 was reduced. We measured the amounts of neo-synthesized PA in roots, and found a decrease in PA only in the lpaat4;lpaat5 double mutant treated with CI976, suggesting that the protein transport impairment was due to a critical PA concentration threshold.

Hydrogen Peroxide Increases during Endodormancy and Decreases during Budbreak in Grapevine (Vitis vinifera L.) Buds

Changes in the level of hydrogen peroxide (H2O2) is a good indicator to monitor fluctuations in cellular metabolism and in the stress responses. In this study, the changes in H2O2 content during bud endodormancy (ED) and budbreak were analysed in grapevine (Vitis vinifera L.). The results showed a gradual increase in the H2O2 content during the development of bud ED, which was mainly due to an increase in the activity of peroxidases (PODs). The maximum H2O2 content reached in the grapevine buds coincided with the maximum depth of bud ED. In contrast, during budbreak, the H2O2 content decreased. As the plant hormones cytokinin (CK) and auxin play an important role in budbreak and growth resumption in grapevine, the effect of exogenous applications of H2O2 on the expression of genes involved in CK and auxin metabolism was analysed. The results showed that H2O2 represses the expression of the CK biosynthesis genes VvIPT3a and VvLOG1 and induces the expression of the CK-inactivating gene VvCKX3, thus reducing potentially the CK content in the grapevine bud. On the other hand, H2O2 induced the expression of the auxin biosynthesis genes VvAMI1 and VvYUC3 and of the auxin transporter gene VvPIN3, thus increasing potentially the auxin content and auxin transport in grapevine buds. In general, the results suggest that H2O2 in grapevine buds is associated with the depth of ED and negatively regulates its budbreak.

Analysis of Graviresponse and Biological Effects of Vertical and Horizontal Clinorotation in Arabidopsis thaliana Root Tip

Clinorotation was the first method designed to simulate microgravity on ground and it remains the most common and accessible simulation procedure. However, different experimental settings, namely angular velocity, sample orientation, and distance to the rotation center produce different responses in seedlings. Here, we compare A. thaliana root responses to the two most commonly used velocities, as examples of slow and fast clinorotation, and to vertical and horizontal clinorotation. We investigate their impact on the three stages of gravitropism: statolith sedimentation, asymmetrical auxin distribution, and differential elongation. We also investigate the statocyte ultrastructure by electron microscopy. Horizontal slow clinorotation induces changes in the statocyte ultrastructure related to a stress response and internalization of the PIN-FORMED 2 (PIN2) auxin transporter in the lower endodermis, probably due to enhanced mechano-stimulation. Additionally, fast clinorotation, as predicted, is only suitable within a very limited radius from the clinorotation center and triggers directional root growth according to the direction of the centrifugal force. Our study provides a full morphological picture of the stages of graviresponse in the root tip, and it is a valuable contribution to the field of microgravity simulation by clarifying the limitations of 2D-clinostats and proposing a proper use.

Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing

Auxin is a key regulator of plant growth and development. Local auxin biosynthesis and intercellular transport generates regional gradients in the root that are instructive for processes such as specification of developmental zones that maintain root growth and tropic responses. Here we present a toolbox to study auxin-mediated root development that features: (i) the ability to control auxin synthesis with high spatio-temporal resolution and (ii) single-cell nucleus tracking and morphokinetic analysis infrastructure. Integration of these two features enables cutting-edge analysis of root development at single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of auxin biosynthesis. We show directional auxin flow in the root and refine the contributions of key players in this process. In addition, we determine the quantitative kinetics of Arabidopsis root meristem skewing, which depends on local auxin gradients but does not require PIN2 and AUX1 auxin transporter activities. Beyond the mechanistic insights into root development, the tools developed here will enable biologists to study kinetics and morphology of various critical processes at the single cell-level in whole organisms.

Abscisic Acid Regulates the Root Growth Trajectory by Reducing Auxin Transporter PIN2 Protein Levels in Arabidopsis thaliana

The root is in direct contact with soil. Modulation of root growth in response to alterations in soil conditions is pivotal for plant adaptation. Extensive research has been conducted concerning the adjustment of root elongation and architecture in response to environmental factors. However, little is known about the modulation of the root growth trajectory, as well as its hormonal mechanism. Here we report that abscisic acid (ABA) participated in controlling root growth trajectory. The roots upon ABA treatment or from ABA-accumulation double mutant cyp707a1,3 exhibit agravitropism-like growth pattern (wavy growth trajectory). The agravitropism-like phenotype is mainly ascribed to the compromised shootward transportation of auxin since we detected a reduced fluorescence intensity of auxin reporter DR5:VENUS in the root epidermis upon exogenous ABA application or in the endogenous ABA-accumulation double mutant cyp707a1,3. We then tried to decipher the mechanism by which ABA suppressed shootward auxin transport. The membrane abundance of PIN2, a facilitator of shootward auxin transport, was significantly reduced following ABA treatment and in cyp707a1,3. Finally, we revealed that ABA reduced the membrane PIN2 intensity through suppressing the PIN2 expression rather than accelerating PIN2 degradation. Ultimately, our results suggest a pivotal role for ABA in the root growth trajectory and the hormonal interactions orchestrating this process.