Identification and evaluation of suitable reference genes for gene expression analysis in rubber tree leaf.

Gene expression profiles are increasingly applied to investigate molecular mechanism for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber tree, however, little is known in leaf. The main objective of this current study was to identify some stable expression reference genes at various developmental stages of leaf, as well as during abiotic (high and low temperature extremes) and biotic stresses (pathogen stress). Gene expression profilings identified the ubiquitin-proteasome system as excellent potential as reference genes for rubber tree leaf. Among a total of 30 tested genes investigated, 24 new candidate (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new candidate genes had better expression stability comparing with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes for rubber tree leaf under four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data. Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other species.

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE5.

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-related protein kinase3-type protein kinase, SOS2-like protein kinase5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, abscisic acid-insensitive5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-like calcium binding proteins) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana).

HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.

Transcriptome analysis reveals the molecular mechanisms of rubber biosynthesis and laticifer differentiation during rubber seed germination.

The molecular mechanisms underlying the initiation of natural rubber synthesis and laticifer differentiation have not been fully elucidated. In this study, we conducted a time-series transcriptome analysis of five rubber tree tissues at four stages of seed germination. A total of 161,199 DEGs were identified between the two groups, including most 16,673 DEGs (A3 vs B3 and A3 vs C3) and lest 1,210 DEGs (C2 vs D2). We found that the maturation of the seed is accompanied by the formation of laticifer cells in cotyledon. Meanwhile, the analysis of hormones related genes expression may provide effective clues for us to promote the differentiation of laticifer cells in seeds by hormones in the future. In this study, hormone-related gene enrichment analyses revealed that IAA, GA, and CTK were activated in laticifer containing tissues. Similarly, GO and GEGG analysis showed that hormone pathways, especially the auxin pathway, are enriched. Gene expression clustering was analyzed using the short time-series expression miner (STEM), and the analysis revealed four distinct trends in the gene expression profiles. Moreover, we enriched transcription factor (TF) enrichment in cotyledon and embryonic axis tissues, and the MYB type exhibited the most significant difference. Furthermore, our findings revealed that genes related to rubber synthesis exhibited tissue-specific expression patterns during seed germination. Notably, key genes associated with rubber biosynthesis, specifically small rubber particle protein (SRPP) and cis-prenyltransferase (CPT), exhibited significant changes in expression in cotyledon and embryonic axis tissues, suggesting synchronous rubber synthesis with seed germination. Our staining results reveled that laticifer cells were exits in the cotyledon before seed imbibition stage. In conclusion, these results lay the foundation for exploring the molecular mechanisms underlying laticifer differentiation and rubber synthesis during seed germination, deepening our understanding of the initiation stages of rubber biosynthesis and laticifer differentiation.

The Arabidopsis chaperone J3 regulates the plasma membrane H+-ATPase through interaction with the PKS5 kinase.

The plasma membrane H(+)-ATPase (PM H(+)-ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H(+)-ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H(+)-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H(+)-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H(+)-ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H(+)-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H(+)-ATPase activity by J3 takes place via inactivation of the PKS5 kinase.

The Calcium Sensor Calcineurin B-Like Proteins -Calcineurin B-Like Interacting Protein Kinases Is Involved in Leaf Development and Stress Responses Related to Latex Flow in Hevea brasiliensis.

Latex flow in Hevea brasiliensis (the Para rubber tree), the sole commercial source of natural rubber (cis-1,4-polyisoprene, NR), renders it uniquely suited for the study of plant stress responses. Calcineurin B-like interacting protein kinases (CIPK) serving as calcium-sensor protein kinases react with calcineurin B-like proteins (CBL) to play crucial roles in hormone signaling transduction and response to abiotic stress in plant developmental processes. However, little is known about their functions in Hevea. In this study, a total of twelve CBL (HbCBL) and thirty CIPK (HbCIPK) genes were identified from the Hevea genome. Structure and phylogenetic analysis assigned these CIPKs to five groups and CBLs to four groups, and mapped onto fourteen of the eighteen Hevea chromosomes. RNA-seq and qPCR analysis showed that the expressions of HbCBL and HbCIPK genes varied in the seven Hevea tissues examined, i.e., latex (cytoplasm of rubber-producing laticifers), bark, leaf, root, seed, female flower, and male flower. The expressions of two HbCBL and sixteen HbCIPK genes showed upward trends during leaf development. Following ethylene yield stimulation and the latex tapping treatment, both practices invoking stress, the expression levels of most latex-expressed genes were significantly altered. Yeast two-hybrid test revealed interactions for multiple combinations of HbCBLs and HbCIPKs with substantial gene expression in latex or other Hevea tissues. However, all the HbCBL-HbCIPK complexes examined did not recruit HbSOS1 or AtSOS1 to form functional salt tolerance SOS pathway in yeast cells. Taken together, the results suggested a role of the Hevea CBL-CIPK network as a point of convergence for several different signaling pathways in growth, development, and stress responses in relation to latex production.

The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree).

Efficient sucrose loading in rubber-producing cells (laticifer cells) is essential for retaining rubber productivity in Hevea brasiliensis, but the molecular mechanisms underlying the regulation of this process remain unknown. Here, we functionally characterized a putative Hevea SUT member, HbSUT3, mainly in samples from regularly exploited trees. When expressed in yeast, HbSUT3 encodes a functional sucrose transporter that exhibits high sucrose affinity with a K(m) value of 1.24 mm at pH 4.0, and possesses features typical of sucrose/H(+) symporters. In planta, when compared to the expression of other Hevea SUT genes, HbSUT3 was found to be the predominant member expressed in the rubber-containing cytoplasm (latex) of laticifers. The comparison of HbSUT3 expression among twelve Hevea tissues demonstrates a relatively tissue-specific pattern, i.e. expression primarily in the latex and in female flowers. HbSUT3 expression is induced by the latex stimulator Ethrel (an ethylene generator), and relates to its yield-stimulating effect. Tapping (the act of rubber harvesting) markedly increased the expression of HbSUT3, whereas wounding alone had little effect. Moreover, the expression of HbSUT3 was found to be positively correlated with latex yield. Taken together, our results provide evidence favouring the involvement of HbSUT3 in sucrose loading into laticifers and in rubber productivity.

Characterization of the rubber tree metallothionein family reveals a role in mitigating the effects of reactive oxygen species associated with physiological stress.

Metallothioneins (MTs) as reactive oxygen species (ROS) scavengers play important roles in stress response and heavy metal homeostasis. In Hevea brasiliensis (the para rubber tree that is the source of commercial natural rubber) and in other trees, the functions of MTs are not well understood. Latex exudes when the rubber tree is tapped. The flow of latex and its regeneration can be enhanced by tapping, wounding and ethylene treatment, all of which produce ROS as a by-product. Here, we show the presence of four MT genes in H. brasiliensis, comprising three Type 2 (HbMT2, -2a and -2b) and one Type 3 (HbMT3L) isoforms, representing one of the smallest MT gene families among angiosperms. The four HbMTs exhibited distinct tissue expression patterns: HbMT2 and HbMT3L mainly in leaves, HbMT2a specifically in flowers and HbMT2b in diverse tissues. The expression of HbMT2b, an isoform present in latex, decreased significantly in the latex following the stress-inducing treatments of tapping, wounding and ethephon (an ethylene generator). The expressions of the leaf-abundant isoforms, HbMT2 and -3L were up-regulated following pathogenic fungus infection and high-temperature stress, but down-regulated by low-temperature stress. These reactions were consistent with multiple defense- and hormone-responsive cis-acting elements in the HbMT promoters. Nine transcription factors were shown to implicate in the high-temperature responsiveness of HbMT2 and -3L in leaves. Overexpression of HbMT2 in Escherichia coli enhanced the bacterium's tolerance to heavy metals and ROS, consistent with its predicted role as an ROS scavenger. Taken together, our results, along with other relevant studies, suggest an important role of HbMTs in latex regeneration as well as species adaptation via the regulation of ROS homeostasis.

The rubber tree genome reveals new insights into rubber production and species adaptation.

The Para rubber tree (Hevea brasiliensis) is an economically important tropical tree species that produces natural rubber, an essential industrial raw material. Here we present a high-quality genome assembly of this species (1.37 Gb, scaffold N50 = 1.28 Mb) that covers 93.8% of the genome (1.47 Gb) and harbours 43,792 predicted protein-coding genes. A striking expansion of the REF/SRPP (rubber elongation factor/small rubber particle protein) gene family and its divergence into several laticifer-specific isoforms seem crucial for rubber biosynthesis. The REF/SRPP family has isoforms with sizes similar to or larger than SRPP1 (204 amino acids) in 17 other plants examined, but no isoforms with similar sizes to REF1 (138 amino acids), the predominant molecular variant. A pivotal point in Hevea evolution was the emergence of REF1, which is located on the surface of large rubber particles that account for 93% of rubber in the latex (despite constituting only 6% of total rubber particles, large and small). The stringent control of ethylene synthesis under active ethylene signalling and response in laticifers resolves a longstanding mystery of ethylene stimulation in rubber production. Our study, which includes the re-sequencing of five other Hevea cultivars and extensive RNA-seq data, provides a valuable resource for functional genomics and tools for breeding elite Hevea cultivars.

Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree.

In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments.

Molecular characterization and expression analysis of the small GTPase ROP members expressed in laticifers of the rubber tree (Hevea brasiliensis).

ROP (Rho of plants) proteins are plant-specific Rho-type small GTPases which play important roles in cellular processes and stress responses. This study explores the characteristics and possible functions of ROPs that are expressed primarily in laticifers of the rubber tree (Hevea brasiliensis). The work serves as a preliminary step to determining their involvement in latex flow and regeneration, laticifers formation and tapping panel dryness (TPD, a physiological disorder in rubber trees that result in the stoppage of latex flow). In this connection, we (i) identified five HbROPs (HbROP1-HbROP5) by searching latex transcripts database and the genome databases, (ii) characterized molecular and phylogenic aspects of the HbROPs and examined the cis-regulatory elements in their promoter regions; (iii) analyzed by Real-time Quantitative PCR (QPCR) the tissue specificity of the HbROPs and their expression patterns in response to tapping, bark wounding and growth regulator treatments. All five HbROP genes were strongly expressed in the latex, with HbROP1, 3, 4 and 5 showing the highest expression among the six Hevea tissues examined, viz. latex, bud, mature leaf, bark, male flower and seed. When tapping was initiated on previously untapped trees, HbROP3 transcription was substantially down-regulated whereas HbROP5 expression was markedly up-regulated. Transcripts of HbROP3 rose gradually with the development of TPD. Except for the cytokinin 6-benzyl aminopurine that induced a rise in HbROP5 transcripts by more than 2-fold, the other growth regulators tested had little effect on HbROPs expression. The roles of HbROPs in rubber tree are discussed in relation to the diverse functions of ROP homologs reported in other plant species.

Molecular characterization and expression analysis of cDNAs encoding four Rab and two Arf GTPases in the latex of Hevea brasiliensis.

In plants, Rab and Arf GTPases are key regulators of vesicle trafficking. To investigate whether these small GTPases (SG) play a role in the regulation of the regeneration of latex (the cytoplasm of the rubber-producing laticifer cell) in Hevea brasiliensis (Hevea hereafter), full-length cDNAs that encode four HbRab and two HbArf GTPases were cloned. The four HbRab proteins showed specific GTP-binding activity when expressed in Escherichia coli. Transcript expression of the six SG genes was investigated by real-time RT-PCR. All genes revealed to be expressed in each of the six Hevea tissues examined, but the expression patterns were different. Four genes, HbArf1, HbRab2, HbRab3 and HbRab4, displayed a preferential expression in latex. The expression of all genes was upregulated by the act of latex exploitation (tapping), and HbRab1 had the highest level of upregulation. Wounding markedly upregulated the expression of two SG genes (HbRab1 and HbArf2), and exogenous methyl jasmonate upregulated all six SG genes. Wounding might upregulate the expression of HbRab1 and HbArf2 through a jasmonic acid-mediated signaling pathway. None of the genes were markedly upregulated by Ethrel (an ethylene releaser and latex stimulator); instead, HbArf2 and HbRab4 were downregulated significantly after a 24 h treatment with Ethrel. This paper gives the first description of Rab and Arf GTPases in Hevea and provides clues for their involvement, HbRab1 in particular, in latex regeneration.

Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3.

Real-time RT-PCR (RT-qPCR) is a sensitive and precise method of quantifying gene expression, however, suitable reference genes are required. Here, a systematic reference gene screening was performed by RT-qPCR on 22 candidate genes in Hevea brasiliensis. Two ubiquitin-protein ligases (UBC2a and UBC4) were the most stable when all samples were analyzed together. A mitosis protein (YLS8) and a eukaryotic translation initiation factor (eIF1Aa) were the most stable in response to tapping. UBC2b and UBC1 were the most stable among different genotypes. UBC2b and a DEAD box RNA helicase (RH2b) were the most stable across individual trees. YLS8 and RH8 were most stably expressed in hormone-treated samples. Expression of the candidate reference genes varied significantly across different tissues, and at least four genes (RH2b, RH8, UBC2a and eIF2) were needed for expression normalization. In addition, examination of relative expression of a sucrose transporter HbSUT3 in different RNA samples demonstrated the importance of additional reference genes to ensure accurate quantitative expression analysis. Overall, our work serves as a guide for selection of reference genes in RT-qPCR gene expression studies in H. brasiliensis.